JP7315950B2 - Histological detection method and kit for phosphorylated protein - Google Patents

Description

The present invention relates to histological detection methods and kits for phosphorylated proteins.

Alzheimer's disease (AD) is one of dementias that causes memory impairment due to loss and dysfunction of nerve cells in the brain. Pathological features of AD include senile plaques and neurofibrillary tangles (NFTs). Neuritic plaques are formed by deposition of a peptide called Aβ outside nerve cells. The major component of NFTs is tau. Tau is a member of the microtubule-associated protein family, and is physiologically phosphorylated to regulate functions such as stabilization of microtubule polymerization and structural maintenance of the cytoskeleton. NFTs are obtained by polymerizing tau in an abnormally phosphorylated state to form inclusion bodies in nerve cells (Non-Patent Document 1). Thus, diseases associated with accumulation of tau are collectively called tauopathy, and AD is a representative disease of tauopathy. The pathogenesis of AD is still largely unknown, and no fundamental treatment has been established. Therefore, in order to develop an effective therapeutic method, it is urgent to clarify the whole mechanism of tauopathic neurodegeneration.

At present, more than 40 tau phosphorylation sites have been confirmed (Non-Patent Document 2), and it is believed that tau exists in various phosphorylation states in the brain from normal to abnormal tau accumulation (non-patent document 2). Patent document 3). Such phosphorylated tau can be easily detected by biochemical techniques such as Western blotting and ELISA using specific antibodies. However, when the tissue localization of phosphorylated tau is analyzed by methods such as immunohistochemical staining using these antibodies, although aggregated and accumulated tau in the pathological brain may be detected, phosphorylation in normal neurons Unaggregated abnormally phosphorylated tau, which is assumed to be a pre-stage of tau or tauopathic neurodegeneration, is extremely poorly stained. As a result, only NFTs are selectively stained when pathological brain tissue is stained with an anti-phosphorylated tau antibody such as AT8 (Thermo Fisher Scientific Inc.). The cause of this is unknown, and no effective staining method has been established so far. On the other hand, although there are several paper reports that attempted tissue staining of non-aggregated phosphorylated tau (Non-Patent Documents 4, 5, 6), none of them have sufficient sensitivity for wide application, It is difficult to say that the staining image is unexplainable from the original localization of the axons, or that the staining is sufficiently reliable. In addition, the cause of this is unknown although a special structure of aggregated phosphorylated tau is assumed (Non-Patent Document 7), and an effective staining method has not been established. Therefore, the tissue localization of non-aggregated phosphorylated tau leading to the formation of phosphorylated tau or tauopathy in normal brain has not been elucidated.

Hypothermia model by anesthesia is known as a method for artificially accelerating tau phosphorylation in the mouse brain (Non-Patent Document 5). This tau phosphorylation is readily detectable by biochemical techniques. However, this detection is extremely difficult with general histological analysis. In the present invention, a method for histological detection of physiological phosphorylated tau was established using a tau hyperphosphorylation model.

GSK3β is one of the serine threonine protein kinases. Its enzymatic activity is regulated by its own phosphorylation by insulin receptor signals and the like. Since it is ubiquitously expressed in tissues and has a wide variety of substrates, it is involved in a wide range of biological reactions, such as glucose metabolism, neuronal cell development and function. Tau is one of its substrates, and GSK3β is thought to be deeply involved in tau abnormalities and neurodegeneration in Alzheimer's disease. Therefore, detection of phosphorylation that can determine the activity of GSK3β is required in a wide range of research fields. ERK1/2 are a subfamily of Mitogen-activated Protein Kinases (MAPK) that are activated by growth factors such as EGF, cytokines, and oxidative stress. It has been considered to be involved in cell proliferation, division, and cell differentiation, and its relationship with synaptic plasticity in the nervous system has been pointed out. Synaptic plasticity occurs when NMDA-type glutamate receptors are activated in the mouse hippocampus. At this time, ERK1/2 is activated in neurons. type receptor-dependent long-term potentiation is suppressed. ERK1/2 activity is also regulated by phosphorylation by upstream kinases, and the relationship between phosphorylation and ERK activity and various physiological phenomena has been extensively studied. In the present invention, a histological detection method for phosphorylated GSKβ and phosphorylated ERK1/2 was established.

Xie S, Miyasaka T. Role of tau protein in dementia. Basic Aging Research. 2015 39(3):13-22 Iqbal K, Liu F, Gong CX. Tau and neurodegenerative disease: the story so far. Nat Rev Neurol. 2016 12(1):15-27 Miyasaka T, Takashima A. Molecular modification of tau protein. Brain and Nerve. 2002 59(9): 753-766 Planel E, Tatebayashi Y, Miyasaka T, Liu L, Wang L, Herman M, Yu WH, Luchsinger JA, Wadzinski B, Duff KE, Takashima A. Insulin dysfunction induces in vivo tau hyperphosphorylation through distinct mechanisms. J Neurosci. 2007 27( 50): 13635-13648. Planel E, Krishnamurthy P, Miyasaka T, Liu L, Herman M, Kumar A, Bretteville A, Figueroa HY, Yu WH, Whittington RA, Davies P, Takashima A, Nixon RA, Duff KE. Anesthesia-induced hyperphosphorylation detaches 3-repeat tau from microtubules without affecting their stability in vivo. J Neurosci. 2008 28(48):12798-12807. Tuerde D, Kimura T, Miyasaka T, Furusawa K, Shimozawa A, Hasegawa M, Ando K, Hisanaga SI. Isoform-independent and -dependent phosphorylation of microtubule-associated protein tau in mouse brain during postnatal development. J Biol Chem. 2018 293 (5): 1781-1793. Trojanowski JQ, Schuck T, Schmidt ML, Lee VM. Distribution of tau proteins in the normal human central and peripheral nervous system. J Histochem Cytochem 1989 37(2): 209-215

An object of the present invention is to provide a histological detection method for phosphorylated proteins.

a cryosectioning step of preparing a frozen section of a tissue containing a phosphorylated protein; a fumigation step of treating the frozen section with vapor of a tissue fixative to fumigate the tissue; The method comprises a pretreatment step of impregnating a tissue with an ionic surfactant, and a staining step of staining the tissue impregnated with the ionic surfactant with an antibody.

According to the present invention, phosphorylated proteins can be clearly detected histologically, and in particular, non-aggregated phosphorylated tau can be clearly detected histologically. and diagnostically useful.

FIG. 1 is a photographic diagram showing paraffin sections prepared from normothermic (A, B) and hypothermic (C, D) mice and immunohistologically stained with AT8. FIG. 4 shows the state of tau phosphorylation in sections prepared by the fresh freezing method. (A) Fresh frozen sections obtained from normothermic and hypothermic mouse brains were solubilized and subjected to Western blotting. (B) is a diagram quantifying the brightness of the bands recognized by each antibody. FIG. 10 is a diagram showing the results of examination of fixation methods after preparation of fresh frozen sections. (A) and (B) are immersion methods using 4% paraformaldehyde (hereinafter sometimes abbreviated as PFA: ParaFormAldehyde), and (C) and (D) are fumigation methods using 4% paraformaldehyde. is. FIG. 2 shows detection of dephosphorylated tau using a Tau1 antibody by the tissue staining method of the present invention. FIG. 1 is a diagram examining the importance of sodium dodecyl sulfate (hereinafter sometimes abbreviated as SDS: Sodium Dodecyl Sulfate) in pretreatment for immunostaining. (A) Antibody staining with AT8 without treatment, (B) Antibody staining with AT8 after treatment with sodium dodecyl sulfate sample buffer (SDS, containing 2-mercaptoethanol), (C) After treatment with sample buffer without sodium dodecyl sulfate alone FIG. 10 shows the results of antibody staining with AT8 and (D) antibody staining with AT8 after treatment with a sample buffer from which only 2-mercaptoethanol was removed. FIG. 10 is a diagram showing a comparative study of denaturants used for tissue staining pretreatment. (A) 1% sodium dodecyl sulfate, (B) 1% N-lauroyl sarcosinate sodium (hereinafter sometimes referred to as Sarkosyl), (C) RIPA buffer, (D) 8M Urea, (E) 2M Urea, (F) Antibody staining with AT8 after treatment with 6M Guanidin HCl. It is a diagram using the histological detection method of the present invention for other than phosphorylated tau, (A) a photographic diagram of phosphorylated GSK3β by Western blotting, (B) a photographic diagram of phosphorylated GSK3β by antibody staining, ( C) Western blotting photographic representation of phosphorylated ERK1/2, (D) Antibody staining photographic representation of phosphorylated ERK1/2.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. The embodiments are intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is as follows. The scope of the present invention is not limited to the embodiments, and other embodiments in which the configurations of the following embodiments are appropriately replaced by those skilled in the art are also included in the scope of the present invention.

The histological detection method for phosphorylated proteins according to this embodiment includes the following steps (i), (ii), (iii), and (iv).

(i) Cryosectioning step of preparing frozen sections of tissues containing phosphorylated proteins
(ii) a fumigation step in which the frozen sections are treated with tissue fixative vapor to fumigate the tissue;
(iii) a pretreatment step of impregnating the fumigated tissue with an ionic surfactant before staining;
(iv) Antibody staining step of antibody staining the tissue impregnated with the ionic surfactant

Further, the kit for histological detection of phosphorylated proteins according to the present embodiment includes a tissue fixative for treating frozen sections of tissues containing phosphorylated proteins with steam and fumigating the tissues, and and an ionic surfactant with which the tissue is impregnated as a pretreatment, and an antibody staining agent that stains the tissue impregnated with the ionic surfactant with an antibody.

In the frozen section preparation step, a frozen section of the tissue containing the phosphorylated protein is prepared. Phosphorylation of proteins plays an important role in the regulation of cell growth, enzyme activity, intracellular signal transduction, and the like. , phosphorylated tau (phosphorylated tau protein), GSK3β, ERK1/2, etc., preferably phosphorylated tau. Tau protein is present in large amounts in central neurons and is essential for the function of nerve axons that make up the neural network of the brain. Instead, it causes death of nerve cells. A pathological image in which tau protein is fibrilized and deposited in the cytoplasm is called neurofibrillary tangles. This neurofibrillary tangle is observed not only in Alzheimer's disease but also in degenerative diseases such as progressive supranuclear palsy, corticobasal degeneration, frontotemporal dementia, and boxer's encephalopathy. In familial frontotemporal dementia (FTDP), mutations in the tau gene promote the formation of neurofibrillary tangles (NFTs), leading to cognitive symptoms.

In addition, the method for histological detection of phosphorylated proteins according to the present embodiment can detect not only aggregated phosphorylated tau, but also non-aggregated phosphorylated tau. Since neurofibrillary tangles themselves do not show neurotoxicity and the state of tau prior to fibrosis is thought to be the cause of toxicity and memory impairment, the present invention can detect non-aggregated phosphorylated tau as well. Advantages are great.

The method of preparing a frozen section of tissue is not particularly limited. Cut into 10 µm slices.

Next, in the fumigation step, the prepared frozen sections are treated with tissue fixative vapor to fumigate the tissue. The tissue fixative is not particularly limited as long as it has a chemical processing ability to protect the tissue sample from deterioration due to autolysis or putrefaction. For example, formalin fixative with formaldehyde or paraformaldehyde, ethanol, methanol, Alcohol fixatives with acetone or chloroform, or picric acid fixatives such as Bouin's solution can be used, preferably fumigation fixation with formalin vapor with paraformaldehyde. In the case of formalin vapor, fumigation is performed with, for example, 20 g/L to 60 g/L paraformaldehyde, preferably 30 g/L to 50 g/m paraformaldehyde, more preferably 40 g/L paraformaldehyde. For the treatment with formalin vapor, for example, a frozen section sample placed on a sample stage is placed in a closed container, gaseous formalin volatilized by heat is filled in the closed container, and the sample is placed in gaseous formalin. It is a treatment of exposure to

Next, in the pretreatment step, the fumigated tissue is impregnated with an ionic surfactant prior to antibody staining. The ionic surfactant is not particularly limited, and any of an anionic surfactant, a cationic surfactant and an amphoteric surfactant can be used, but an anionic surfactant is preferred.

Anionic surfactants are, for example, sodium lauryl sulfate, sodium lauroyl sarcosinate, sodium stearate, zinc stearate, calcium stearate, magnesium stearate, aluminum stearate, aluminum isostearate, triethanolamine stearate, potassium palmitate, cetyl sodium sulfate, sodium lauryl phosphate, disodium lauryl phosphate, triethanolamine palmitate, sodium polyoxyethylene lauryl phosphate, sodium N-acyl glutamate, sodium palmitate, sodium laurate, potassium laurate, zinc laurate, Triethanolamine Laurate, Sodium Laurate, Lithium Lauryl Sulfate, Potassium Lauryl Sulfate, Ammonium Lauryl Sulfate, Monoethanolamine Lauryl Sulfate, Diethanolamine Lauryl Sulfate, Triethanolamine Lauryl Sulfate, Sodium Lauryl Diaminoethylglycinate, Triethanolamine Alkyl Sulfate Ether, funnel oil, linadodecyl benzene sulfate, polyoxyethylene hydrogenated castor oil maleic acid, acyl methyl taurine, fatty acid soap, α-acyl sulfonate, alkyl sulfonate, alkyl allyl sulfonate, alkyl naphthalene sulfonate, alkyl sulfuric acid salt, POE alkyl ether sulfate, alkyl amide sulfate, alkyl phosphate, POE alkyl phosphate, alkyl amide phosphate, alkyloyl alkyl taurate, N-acyl amino acid salt, POE alkyl ether carboxylate, alkyl Sulfosuccinate, sodium alkylsulfoacetate, acylisethionate, acylated hydrolyzed collagen peptide salt, perfluoroalkyl phosphate, soap base, potash soap, zinc undecylenate, potassium-containing soap base, potassium coconut oil fatty acid, Sulfated castor oil, sodium myristate, potassium myristate, zinc myristate, magnesium myristate, sodium oleate, potassium oleate, triethanolamine N-acyl-L-glutamate, sodium N-acyl-L-glutamate, N -coconut fatty acid acyl-L-glutamate sodium, N-coconut fatty acid/hydrogenated tallow fatty acid acyl-L-glutamate sodium, N-coconut fatty acid acyl-L-glutamate triethanolamine, stearoyl-L-glutamate disodium, N - hydrogenated tallow fatty acid acyl-L-glutamate sodium, N-lauroyl-L-glutamate sodium, N-stearoyl-L-glutamic acid, N-lauroyl-L-lysine, lauroyl-L-glutamic acid triethanolamine, L-arginine ethyl. DL-pyrrolidone carboxylate, N-cocoate acyl-L-arginine ethyl/DL-pyrrolidone carboxylate, cocoate sarcosine, cocoate sarcosine sodium, cocoate sarcosine triethanolamine, lauroyl sarcosine, lauroyl sarcosine Triethanolamine, carboxylated polyoxyethylene tridecyl ether sodium salt (3E. O. ), sodium β-laurylaminopropionate, laurylaminopropionic acid solution, and the like.

In addition, potassium cocoate methyltaurate, sodium cocoate methyltaurate, sodium lauroylmethyltaurate, sodium lauryl sulfoacetate, sodium cocoate methylalanine liquid, triethanolamine cocoate liquid, sodium lauroylmethyl-β-alanine liquid , lauryl-N-carboxymethoxyethyl-N-carboxymethylimidazolinium disodium dodecanoyl sarcosine, coconut oil alkyl-N-carboxyethyl-N-hydroxyimidazolinium betaine sodium, coconut oil alkyl-N-carboxyethoxyethyl- N-carboxyethylimidazolinium disodium hydroxide, coconut alkyl-N-carboxymethoxyethyl-N-carboxymethylimidazolinium disodium hydroxide, coconut alkyl-N-carboxymethoxyethyl-N-carboxymethylimidazoline sodium disodium lauryl sulfate, myristoylmethyl-β-alanine sodium solution, sodium stearoylmethyltaurate, sodium tetradecenesulfonate, sodium dodecylbenzenesulfonate, triethanolamine dodecylbenzenesulfonate, sodium alkanesulfonate, ammonium alkyl sulfate, octylphenoxy Sodium diethoxyethylsulfonate, magnesium coconut oil sulfate/triethanolamine, sodium myristyl sulfate, sodium cetostearyl sulfate, sodium oleyl sulfate, triethanolamine oleyl sulfate, sodium polyoxyethylene lauryl ether sulfate, ammonium polyoxyethylene lauryl ether sulfate (2E.O.), polyoxyethylene lauryl ether sulfate triethanolamine, polyoxyethylene alkyl ether ammonium sulfate (3E.O.) liquid, polyoxyethylene alkyl ether sulfate diethanolamine (3E.O.) liquid, polyoxyethylene alkyl triethanolamine ether sulfate (3E.O.) liquid, sodium polyoxyethylene alkyl ether sulfate (3E.O.) liquid, hydrogenated coconut oil fatty acid glyceryl sodium sulfate, polyoxyethylene (5) coconut oil fatty acid monoethanolamide phosphoric acid Ester, Polyoxyethylene Alkyl (12-14) Ether Phosphate (2E.O.), (8E.O.), (10E.O), Polyoxyethylene Lauryl Ether Phosphate, Sodium Polyoxyethylene Lauryl Ether Phosphate , polyoxyethylene cetyl ether phosphate, polyoxyethylene cetyl ether sodium phosphate, polyoxyethylene stearyl ether, polyoxyethylene cholesteryl ether, polyoxyethylene stearyl ether phosphate, polyoxyethylene oleyl ether phosphate, polyoxyethylene oleyl Sodium ether phosphate, polyoxyethylene oleyl ether diethanolamine phosphate, polyoxyethylene alkylphenyl ether phosphate, polyoxyethylene alkylphenyl ether phosphate triethanolamine, polyoxyethylene polyoxypropylene cetyl ether phosphate, polyoxypropylene glyceryl Ether phosphoric acid, polyoxypropylene butyl ether phosphate, polyoxyethylene lauryl disodium sulfosuccinate, coconut oil fatty acid ethyl ester sodium sulfonate, dioctyl sodium sulfosuccinate, octylphenoxydiethoxyethyl sulfonate sodium, lauryl disodium sulfosuccinate, olein acid amide disodium sulfosuccinate, polyoxyethylene alkyl (C12-15) ether phosphate, polyoxyethylene alkyl (C12-14) disodium sulfosuccinate (7E. O. ), disodium polyoxyethylene lauroyl ethanolamide sulfosuccinate (5E.O.), sodium isostearoyl lactylate, undecylenoyl hydrolyzed collagen sodium, undecylenoyl hydrolyzed collagen potassium, undecyl hydroxyethylimidazolinium betaine sodium, lauric acid hydrolyzate Collagen sodium, coconut oil fatty acid hydrolyzed collagen sodium, coconut oil fatty acid hydrolyzed collagen potassium, coconut oil fatty acid hydrolyzed collagen potassium liquid, coconut oil fatty acid hydrolyzed collagen triethanolamine, isostearoyl hydrolyzed collagen, isostearoyl hydrolyzed silk, isostearoyl hydrolyzed collagen aminomethylpropanediol salt, ethylenediamine-N,N,N',N'-tetrakis(2-hydroxypropyl)diolate, dodecanoyl sarcosine, sodium laurylaminodipropionate solution (30%), Oleoyl sarcosine, myristoyl-beta-alanine sodium solution, undecylhydroxyethylimidazolinium betaine sodium, alkyl (C12,13,16) ammonium sulfate, alkyl (C11,13,15) triethanolamine sulfate (1), alkyl ( C11,13,15) Triethanolamine Sulfate (2), Alkyl (C12-15) Triethanolamine Sulfate, Alkyl (C12-14) Triethanolamine Sulfate, Alkyl (12,13) Sodium Sulfate, Polyoxyethylene Alkyl Ether Triethanolamine sulfate (3E.O.) liquid, (C11-15) sodium pareth-3 sulfate, (C12,13) sodium pareth-3 sulfate, (C12-15) sodium pareth-3 sulfate, (C12,13) Paleth-3 Sulfate TEA, (C12,13) Palace-3 Sulfate (TEA/Na), PEG-3 Coconut Amide MEA Sodium Sulfate, PEG-5 Coconut Amide MEA Phosphate, PEG-5 Lauryl Citrate Sulfosuccinate Disodium , PEG-5 ceteth-10 phosphate, PEG-25 butyl phosphate, sodium alkyl (C14-18) sulfonate, alkyl (C20-22) phosphate, isolaureth-4 phosphate, undecylenoylglycine, potassium olivate, Oleyl sarcosine, oleth methyl taurate sodium, oleth-3 phosphate, oleth-4 phosphate, oleth-5 phosphate, oleth-10 phosphate, oleth-20 phosphate, oleth-7 sodium phosphate, oleth-8 phosphate Sodium, sodium olefin (C14-16) sulfonate, capryloylglycine, sodium cocoylamino acid, cocoylalanine triethanolamine, sodium cocoyl isethionate, ammonium cocoyl isethionate, potassium cocoylglycinate, sodium cocoylglycinate, cocoyl glutamine triethanol Amines, cocoyl glutamates such as cocoyl glutamic acid, disodium cocoyl glutamate, potassium cocoyl glutamate, cocoyl sarcosine, cocoyl sarcosine sodium, cocoyl sarcosine triethanolamine, sodium cocoyl taurate, cocoyl methyl alanine, cocoyl methyl alanine sodium, cocoyl methyl taurine, cocoyl Potassium Methyl Taurate, Magnesium Cocoyl Methyl Taurate, Sodium Cocoyl Methyl Taurate, Sodium Cocoyl Malo Amino Acid, Sodium Coceth Sulfate, Di(C11-15) Pareth-2 Phosphate, Di(C12-15) Pareth-4 Phosphate, Di(C12) -15) pareth-8 phosphate, di(C12-15) pareth-10 phosphate, dioleyl phosphate, dioleth-8 sodium phosphate, dioleth-8 sodium phosphate, dicocoylethylenediamine PEG-15 sodium sulfate, dilaureth-10 Sodium phosphate, steareth-2-phosphate, steareth-3-phosphate, calcium stearoyl lactylate, sodium stearoyl lactylate, undecylenamide sulfosuccinate MEA-2Na, lauramide sulfosuccinate MEA-2Na, disodium laureth sulfosuccinate, DEA cetyl phosphate, cetyl phosphate Potassium, Sodium Cetearyl Sulfate, Ceteth-10 Phosphate, Ceteth-20 Phosphate, Triceteareth-4 Phosphate, Tricedes-4 Carboxylic Acid, Tricedes-8 Carboxylic Acid, Sodium Tricedes-4 Carboxylate, Sodium Tricedes-7 Carboxylate, Tricedes-3 Sodium Acetate, Tricedes-7 Potassium Phosphate, Trilaureth-4 Phosphate, Oleyl Lactate, Sodium Palmate Glutamate, Magnesium Palmitoyl Glutamate, Sodium Palmitoyl Sarcosinate, Palmitoyl Proline, Sodium Palmitoyl Proline, Sodium Palmitoyl Methyl Taurate, Potassium Myristoyl Glutamate , sodium myristoyl glutamate, sodium myristoyl sarcosinate, sodium myristoyl methyl taurate, potassium cocoate, triethanolamine cocoate, arginine cocoate, sodium lauraminodiacetate, sodium lauryl glycol acetate, laureth-5 carboxylic acid, laureth-6 carboxylic acid, laureth -11 carboxylic acid, sodium laureth-5 carboxylate, sodium laureth-6 carboxylate, sodium laureth-11 carboxylate, laureth-5 acetic acid, laureth-6 acetic acid, potassium laureth-4,5 acetate, sodium laureth-3 acetate, Sodium Laureth-4 Acetate, Sodium Laureth-5 Acetate, Sodium Laureth-6 Acetate, Sodium Laureth-11 Acetate, MIPA Laureth Sulfate, Sodium Laureth Sulfate, Triethanolamine Laureth Sulfate, Ammonium Laureth-2 Sulfate, Ammonium Laureth-3 Sulfate, Laureth- monophosphate, laureth-diphosphate, laureth-tetraphosphate, sodium lauroyl aspartate, sodium lauroyl oat amino acid, sodium lauroyl lactate, potassium lauroylmethylalanine, lauroylmethylalanine triethanolamine, dicetyl phosphate, cetyl phosphate, etc. is.

Cationic surfactants include, for example, lauryltrimethylammonium chloride, dicocoyldimethylammonium chloride, myristyldimethylbenzylammonium chloride, cetyltrimethylammonium chloride, laurylpyridinium chloride, cetylpyridinium chloride, benzethonium chloride, stearyltrimethylammonium chloride, benzalkonium chloride. , benzalkonium chloride solution, laurylamine oxide, alkyltrimethylammonium chloride, stearyltrimethylammonium chloride, stearyldimethylbenzylammonium chloride, dimethylstearylammonium chloride-treated hectorite, benzyldimethylstearylammonium chloride-treated hectorite, distearyldimethylammonium chloride, Distearyldimethylammonium chloride-treated bentonite, dialkyldimethylammonium chloride, tri(polyoxyethylene)stearylammonium chloride (5E.O.), di(polyoxyethylene)oleylmethylammonium chloride (2E.O.), stearoylcolamino chloride Formylmethylpyridinium, polyoxypropylenemethyldiethylammonium chloride, alkyldiaminoethylglycine hydrochloride liquid, alkylisoquinolinium bromide liquid, lauryltrimethylammonium bromide, cetyltrimethylammonium bromide, stearyltrimethylammonium bromide, cetyltrimethylammonium saccharin , stearyltrimethylammonium saccharin, polyethylene glycol/epiclurhydrin/coconut oil alkylamine/dipropylenetriamine condensate, polyethylene glycol/epichlorohydrin/beef tallow alkylamine dipropylenetriamine condensate, ethyl sulfate lanolin fatty acid aminopropylethyldimethyl ammonium (1), (2), behenyltrimethylammonium bromide, amidopropyldimethylhydroxypropylammonium chloride beheninate, diethylaminoethylamide stearate, dimethylaminopropylamide stearate, lanolin derivative quaternary ammonium salt, PEG-5 oleamine , PEG-2 oleammonium chloride, PEG-2 cocamine, PEG-3 cocamine, PEG-5 cocamine, PEG-10 cocamine, PEG-15 cocamine, PEG-2 dimedouform amide ethylmonium methosulfate, alkyl (C12,14 ) oxyhydroxypropylarginine hydrochloride, alkyl(C16,18)trimonium chloride, isoalkyl(C10-40)amidopropylethyldimonium ethosulfate, isostearylethylimidazolinium ethosulfate, caesalpinia spinosa hydroxypropyltrimonium chloride, Quaternium-33, Quaternium-91, Cocamidopropyl PG Dimonium Chloridonic Acid, Cocamidopropyl Betainamide MEA Chloride, Cocoyl Arginine Ethyl PCA, Cocotrimonium Methosulfate, Koloha Hydroxypropyltrimonium Chloride, Acetic Acid (Myristamide/Palmitamide) Butylguanidine, dialkyl (C12-18)dimonium chloride, dicocylethylhydroxyethylmonium methosulfate, dicocodimonium chloride, distearyldimonium chloride, distearoylethylhydroxyethylmonium methosulfate, dicetyldimonium chloride, dipalmitoyl Ethyl hydroxyethylmonium methosulfate, dihydroxypropyl PEG-5 linoleammonium chloride, dimethyl PABA amidopropyl laurdimonium tosylate, dimethylstearamine, stearamidoethyldiethylamine, stearamidopropyldimethylamine, stearalkonium chloride, stearyltrimonium saccharin , steartrimonium chloride, steartrimonium bromide, stearoxypropyltrimonium chloride, cetalconium chloride, cetylpyridinium chloride, cetrimonium chloride, cetrimonium bromide, cetrimonium methosulfate, soitrimonium chloride, palmitamidopropyl tri monium chloride, panthenylhydroxypropylsteardimonium chloride, hydroxyethylcetyldimonium phosphate, hydroxypropyltrimonium honey, hydroxypropylbishydroxyethyldimonium chloride, behenamidopropyldimethylamine, behenamidopropyldimethylamine, behenamidopropyldimethylamine, Hentrimonium chloride, behentrimonium methosulfate, poloxamine 701, poloxamine 702, poloxamine 704, bishydroxyethyl cetylamide malonate, coconut alkyl PG dimonium chloride sodium phosphate, lauryltrimonium chloride, laurylpyridinium chloride, linol Amidopropyl PG dimonium chloride phosphate and the like.

Amphoteric surfactants are, for example, lauryldimethylaminoacetic acid betaine, 2-alkyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine, undecyl-N-hydroxyethyl-N-carboxymethylimidazolium betaine, coconut oil alkyl Betaine, coco fatty acid amidopropyl betaine, stearyldihydroxyethyl betaine, stearyldimethylaminoacetate betaine, bis(stearyl-N-hydroxyethylimidazoline) chloroacetic acid complex, coconut oil fatty acid hydrolyzed collagen, oleoyl hydrolyzed collagen, hydrolyzed collagen Hexadecyl, lauramidopropyl betaine solution, myristoyl hydrolyzed collagen solution, alkylaminoethylglycine hydrochloride solution, carboxybetaine type such as lecithin, amidobetaine type, sulfobetaine type, hydroxysulfobetaine type, amidosulfobetaine type, phosphobetaine type, aminocarboxylate type, imidazoline derivative type, amidoamine type, PEG-3 lauramine oxide, oleamine oxide, oleyl betaine, (capryl/capramido)propyl betaine, cocoamine oxide, sodium cocoamphoacetate, sodium cocoamphodiacetate, cocoamphodia Disodium acetate, sodium cocoamphopropionate, dihydroxyethyl lauramine oxide, stearamine oxide, stearyl betaine, palm kernel fatty acid amidoethyl hydroxyethyl aminopropionate sodium, palm kernel fatty acid amidopropyl betaine, hydroxyalkyl (C12,14) hydroxy Ethylalanine, heptadecyl hydroxyethyl carboxylate methylimidazolinium chloride/heptadecyl bishydroxyethylimidazolinium, myristamidopropyl betaine, myristamine oxide, myristyl betaine, lauramidopropylamine oxide, lauramidopropyl hydroxysultaine, laura midopropyl betaine, sodium lauraminopropionate, lauramine oxide, sodium lauriminodipropionate, lauryl hydroxysultaine, lauryl betaine, sodium lauroamphoacetate, lauroyl lysine and the like.

Next, in the antibody staining step, the tissue impregnated with the ionic surfactant is stained with an antibody. Antibody staining is a method of detecting antigens in tissue using antibodies. The specificity of antibodies can be used to detect antigens and observe the localization of antigens. Antibodies used for antibody staining are not particularly limited, but examples of antibodies that bind to tau include Anti-Rat Tau, RTM38, AT8, PHF1, and Tau1.

It is preferable to have a blood removal step of perfusion blood removal before the frozen section preparation step described above. The blood removal step is for suppressing non-specific staining of blood vessels by antibody staining, and does not affect the detection of phosphorylated tau itself. Bleeding can be performed with, for example, physiological saline.

1. Examination until finding essential steps of the present invention (frozen section preparation step, fumigation step, pretreatment step of impregnating with ionic surfactant, and staining step) [Detection of phosphorylated tau by general tissue staining method]
First, we tried to detect phosphorylated tau in the phosphorylated mouse model brain by a general histological analysis method. Normothermic (37°C) and hypothermic (< 30°C) C57BL/6 strain wild-type and tau knockout (TKO) mice (both males and females were used at 10-14 weeks of age). were perfusion-fixed with PBS containing 4% paraformaldehyde, and paraffin-embedded sections were prepared according to a standard method. Hypothermia-induced tau hyperphosphorylation mouse models were created according to the method of Planel et al. Pentobarbital (Somnopentyl: Kyoritsu Seiyaku Co., Ltd.) was administered to mice, and they were left at room temperature while under deep anesthesia. Rectal temperature was measured with a rectal thermometer, and hypothermia was determined when it fell below 30°C (preferably below 25°C).

Paraffin-embedded sections were prepared according to the following procedure. That is, the mice were confirmed to be normothermic (around 37°C) and hypothermic (below 30°C) after anesthesia, and the abdomen was opened to expose the heart. A 27G hypodermic needle (TERUMO) was connected to the tip of a peristaltic pump (ATTO) tube and inserted into the left ventricle of the exposed heart to incise the right atrium. The injection needle inserted into the left ventricle was perfused with PBS from a peristaltic pump, and blood was exsanguinated from the incision site of the right atrium. After that, the infusion solution was changed to 4% paraformaldehyde in PBS, and perfusion fixation was performed in the same manner. After fixation, the mice were decapitated and fixed in 4% paraformaldehyde for 48 hours. After that, the brain was excised and replaced with ethanol, xylene, and warm paraffin to prepare a paraffin-embedded block. Paraffin blocks were sliced at 6 μm using a microtome, attached to glass slides and dried. The prepared paraffin section was immersed in xylene, ethanol and water in that order to hydrate.

Figure 1 shows typical paraffin sections of normothermic (A, B) and hypothermic (C, D) mice, immunized with AT8, a representative anti-phospho-tau antibody, after treatment with Citrate buffer. It is a photographic diagram of tissue staining. A, C are wild-type mice, B, D are tau knockout mice. E is the result of Western blotting validation of tau phosphorylation in hypothermia treatment. Tau-like staining by AT8 was not observed regardless of body temperature or genotype. In addition, after making paraffin sections, fumigation fixation with 4% paraformaldehyde, pretreatment with sodium dodecyl sulfate, and staining with AT8 were attempted, but no phosphorylated tau staining was observed. As shown on the left side of E, there was a clear AT8-positive band in the hypothermic mouse brain. E Right side shows the quantification of the intensity of the bands. Staining with AT8 was significantly (**, p > 0.01) elevated. From the above results, it was found that tau was clearly phosphorylated by hypothermia treatment, but could not be detected histologically.

Although tau exists widely in the cerebral cortex (Cor) and hippocampus (Hp), it is strongly stained in mossy fibers running in the hippocampal CA3 region. Structures consisting of neuronal cell bodies, including the pyramidal cell layer, are poorly stainable. This reflects the axonal localization of tau. Therefore, the presence or absence of accurate staining in the mossy fiber is the criterion for tau staining.

AT8 is an antibody that reacts only when both the 202nd serine and the 205th threonine of tau are phosphorylated, and is widely used to detect tau aggregated in pathological brains. As a result of staining with AT8, almost no staining was observed even in hypothermic mice, and no difference was observed from normothermic wild-type mice or tau knockout mice. The same experiment was performed with another anti-phosphorylated tau antibody, PHF1, except that AT8 was used, but the results were similar for PHF1, and hyperphosphorylation of tau in hypothermia could not be detected. Antigen activation by autoclaving the sections with citrate buffer is generally used to solve the problem of improving immunostaining, but phosphorylated tau cannot be detected by the antibody used in this antigen activation treatment. I didn't. From these results, it was found that it is extremely difficult to detect phosphorylated tau in paraffin sections prepared by a general method. The antibodies used in this example are shown in Table 1 below.

[Quantification of phosphorylated tau until creation of fresh frozen section]
Paraffin sections require complex steps such as fixation, dehydration, clearing with organic solvents, and embedding in warm paraffin before sectioning. In addition, such a section cannot be analyzed by a biochemical method after preparation of the section. On the other hand, slices were prepared by the fresh freezing method in which the excised brain was quickly frozen and thinly sliced. Fresh frozen sections were prepared according to the following procedure. That is, after the normal or hypothermic treatment, the brain was immediately excised from the mouse, floated on a balance dish (BIO-RAD) on liquid nitrogen, and rapidly cooled thereon. After confirming that it was completely frozen, it was stored at -80°C. Using a cryostat (Leica), slices were sliced at a thickness of 15 µm and stored frozen at -80°C. It was confirmed by Western blotting that tau phosphorylation was preserved even after fresh frozen sections (Fig. 2). FIG. 2 shows the state of tau phosphorylation in sections prepared by the fresh freezing method. (A) Fresh frozen sections from normothermic and hypothermic mouse brains were solubilized and subjected to Western blotting using the indicated antibodies. Anti-Rat Tau (BD Bioscience) is a phosphorylation-independent antibody, and compared to normothermic tau, hypothermic tau has an upwardly shifted band, indicating that it is highly phosphorylated. Recognize. In addition, anti-phosphorylated tau antibodies such as AT8 and PHF1 showed strong reactivity in samples derived from hypothermic brains, while antibodies against dephosphorylated tau such as tau1 showed weak reactivity in hypothermic brains. . From these results, it was found that hyperphosphorylation of tau due to hypothermia was preserved in the sections prepared by the fresh freezing method. (B) is a diagram quantifying the brightness of the bands recognized by each antibody (n=3, Mean±SEM, **: p<0.01*: p<0.05). The results show that phosphorylated tau remains from brain excision to fresh frozen sectioning.

[Immunostaining using fresh frozen sections]
Since it was found that phosphorylated tau remained until preparation of fresh frozen sections, we investigated a tissue fixation method. As a result, no staining property was observed in the fixation by immersing the tissue section in 4% paraformaldehyde solution as in perfusion fixation. Therefore, fumigation method using 4% paraformaldehyde solution (4g/mL paraformaldehyde) was performed aiming at milder tissue fixation. That is, three pieces of Prowipe (Elleair) were placed in a glass staining vat, and 30 mL of 4% paraformaldehyde in PBS was absorbed. After heating to 60°C, the prepared fresh frozen sections were lined up and sealed, followed by fumigation and fixation for 30 minutes. Furthermore, in order to approach the conditions for biochemical analysis by Western blotting, we immersed the fumigated tissue in a sodium dodecyl sulfate sample buffer to change the three-dimensional structure of phosphorylated tau, thereby attempting to detect it. Table 2 below shows the composition and modification conditions of the modifier used in this example.

As a result, AT8 staining was observed mainly in mossy fibers in the hippocampal CA3 region in hypothermic mouse brains (Fig. 3). FIG. 3 is a diagram showing the results of examination of fixation methods after preparation of fresh frozen sections. Fresh frozen sections were post-fixed by immersion (A, B) and fumigation (C, D) using 4% paraformaldehyde, followed by tissue staining with AT8 antibody. Comparing normothermic mice (A, C) and hypothermic mice (B, D), when treated with sodium dodecyl sulfate sample buffer after fixation by the immersion method, no staining was observed. When treated with sodium dodecyl sulfate sample buffer after immobilization by fumigation, AT8-positive phosphorylated tau was observed in both mossy fibers (arrow and inset).

In the same way, except for using AT8 antibody, fresh frozen sections were prepared, post-fixed by fumigation with 4% paraformaldehyde, and tissue stained with PHF1 antibody. Phosphorylated tau was recognized in both cases.

Furthermore, except for the use of AT8 antibody, fresh frozen sections were prepared in the same manner, followed by post-fixation by fumigation using 4% paraformaldehyde, followed by tissue staining with Tau1 antibody, an anti-dephosphorylated tau antibody. As a result, tau staining was observed in normothermic sections, and tau staining was observed in hypothermic sections, although the staining was slightly reduced (Fig. 4). That is, after blood removal with PBS, fresh frozen sections were fixed by fumigation with 4% paraformaldehyde, treated with a sodium dodecyl sulfate sample buffer, and tissue stained with a tau1 antibody. In tau1, tau-like staining observed in normothermic mice was attenuated by hypothermia treatment, but dephosphorylated tau was observed in both normothermic and hypothermic mice. FIG. 4 shows the detection of dephosphorylated tau using the Tau1 antibody by the tissue staining method of the present invention. a is a diagram showing staining at normal body temperature, and b is a diagram showing staining at hypothermia.

These results suggest that a combination of fresh frozen sections and fumigation, which is a combination of relatively weak tissue fixation and protein denaturation treatment using sample buffer, is important for accurate analysis of tau phosphorylation in tissue staining. . Since such staining was not reproduced even when frozen sections were prepared after perfusion fixation with 4% paraformaldehyde, it was determined that fresh frozen sections, fumigation fixation, and sample buffer treatment were essential.

[Study of denaturant used for tissue staining pretreatment]
Staining of phosphorylated tau by this method requires treatment with sodium dodecyl sulfate sample buffer, which is commonly used in Western blotting. This sodium dodecyl sulfate sample buffer contains sodium dodecyl sulfate as a surfactant and 2-mercaptoethanol (2-ME) as a reducing agent. In order to determine these effects, solutions were prepared by removing sodium dodecyl sulfate and 2-ME from the sample buffer, and the effect on AT8 staining was verified (Fig. 5).

FIG. 5 is a diagram examining the importance of sodium dodecyl sulfate in pretreatment for immunostaining. Fresh frozen/fumigated sections prepared from hypothermic mice (A) antibody staining with AT8 without treatment, (B) antibody staining with AT8 after sodium dodecyl sulfate sample buffer treatment, (C) sodium dodecyl sulfate alone. FIG. 4 shows the results of antibody staining with AT8 after sample buffer treatment with 2-mercaptoethanol removed, and (D) antibody staining with AT8 after sample buffer treatment with only 2-mercaptoethanol removed. Clear AT8 staining was observed only when treated with sample buffer containing sodium dodecyl sulfate. Sodium dodecyl sulfate sample buffer treatment is essential for staining with AT8 even for fresh frozen sections/sections prepared by fumigation. turned out to be causing

Sodium dodecylsulfate is a representative ionic surfactant, and has a strong surface-active action for solubilization and protein structure denaturation. A number of surfactants and protein denaturants similar to or related to sodium dodecyl sulfate were tested for AT8 staining (Fig. 6).

FIG. 6 is a diagram showing a comparative study of denaturing agents used for tissue staining pretreatment. (A) 1% sodium dodecyl sulfate, (B) 1% N-lauroyl sarcosinate sodium, (C) RIPA buffer, (D) 8M Urea, (E) 2M for fresh frozen/fumigated sections prepared from hypothermic mice Antibody staining with AT8 was performed after treatment with Urea, (F) 6M Guanidin HCl. As a result, ionic surfactants such as sodium dodecylsulfate and sodium N-lauroylsarcosinate were effective, and RIPA buffer containing sodium dodecylsulfate was also effective. On the other hand, protein denaturants such as chaotropic ions such as Urea and Guanidin HCl were not effective. Thus, it was found that a pretreatment step of impregnating the fumigated tissue with an ionic surfactant such as sodium dodecyl sulfate, N-lauroyl sarcosinate sodium, or RIPA buffer is essential before antibody staining.

Based on the above results, the combination of fresh frozen sections and fumigation fixation with immersion in an ionic surfactant as a pretreatment method was confirmed to be useful as a histological detection method for phosphorylated tau. .

2. Test Examples of the Present Invention Test examples of the method for histological detection of phosphorylated proteins of the present invention are described below.

2-1. Bleeding Step After anesthetizing the mouse to be analyzed, it was fixed on a dissecting table and the abdomen was opened to expose the heart. A 27G injection needle was connected to the tip of a tube such as a peristaltic pump and inserted into the exposed left ventricle of the heart to incise the right atrium. Phosphate buffered saline (PBS: 137 mM NaCl, 2.7 mM KCl, 7 mM Na ₂ HPO ₄ , 1.47 mM KH ₂ PO ₄ , pH 7.4) was perfused from a peristaltic pump to an injection needle inserted into the left ventricle, and an incision was made in the right atrium. Blood flowed from the site.

2-2. Step of Creating Frozen Sections After blood removal, the brains were immediately excised and freeze-frozen on a balance dish floated on liquid nitrogen. After confirming that it was completely frozen, it was stored at -80°C. Sections were prepared using a cryostat (Leica), sliced at a thickness of 15 μm, pasted on MAS-coated slide glasses, dried, and frozen at -80°C.

2-3. Fumigation fixation process Place 3 pieces of Prowipe (Elleair) in a glass staining vat, absorb 30 mL of 4% paraformaldehyde, and preheat sufficiently on a hot plate set to 60°C. The fresh frozen sections prepared in 2-2 were arranged here and fixed (fumigation fixation) at 60°C for 30 minutes. After fixation, they were washed with water for 5 minutes.

2-4. Pretreatment step for impregnation with ionic detergents. For fumigated tissues, sodium dodecyl sulfate sample buffer (2% (w/v) sodium dodecyl sulfate, 80 mM Tris pH 6.8, 10% (w/v) Glycerol, 1% (v/v) 2-Mercaptoethanol (2-ME)), or Tris buffered saline containing 1% sodium dodecyl sulfate, 1% sodium N-lauroylsarcosinate (TBS; 50mM Tris-HCl pH7.6, 152mM NaCl) or RIPA buffer (50mM Tris pH7.6, 150mM NaCl, 1% Nonidet P40, 0.5% (w/v) Sodium Deoxycholate, 0.1% (w/v) Sodium dodecyl sulfate) soaked for 5 minutes. Then, after washing with distilled water for 5 minutes, it was washed with TBS for 5 minutes.

2-4. Antibody staining process Tissues were marked with PAPPEN (Daido Sangyo) and blocked with 10% Goat serum in TBS for 60 minutes. After blocking, 1% Bovine serum albumin (BSA) in TBS containing various primary antibodies was added and allowed to react overnight. After that, the cells were washed 4 times for 5 minutes with TBS containing 0.1% Tween20 (TBS-T), and reacted with a secondary antibody diluted with 1% Bovine serum albumin (BSA) in TBS for 2 hours. After the reaction, the cells were washed with TBS-T for 5 minutes x 4 times and reacted with ABC solution (Avidin-Biotin-Complex; VECTOR LABORATORIES) for 30 minutes.

After washing with TBS-T for 5 minutes, color was developed in TBS containing 0.4 mg/mL diaminobenzidine (DAB) and 0.1% hydrogen peroxide. After confirming color development, the cells were immersed in hematoxylin for about 10 seconds to perform nuclear staining. After staining, the cells were washed with water for 5 minutes, immersed in ethanol and xylene for 5 minutes each 3 times, and mounted.

As shown in Test Example 1, no staining of phosphorylated tau was observed even when frozen sections were prepared after perfusion fixation with 4% paraformaldehyde. In addition, no staining of phosphorylated tau was observed even after antibody staining after fumigation fixation and pretreatment after preparation of paraffin sections. As shown in Test Examples 4, 9, 10 and 11, no staining of phosphorylated tau was observed even with antibody staining in the cryosection preparation and fumigation-fixing steps alone. As shown in FIG. 3, no staining of phosphorylated tau was observed even with antibody staining in the cryosection preparation and pretreatment steps alone. However, as shown in Test Examples 2, 3, 5, 6, 7, and 8, staining of phosphorylated tau was clearly observed by antibody staining after fumigation fixation and pretreatment steps after preparation of frozen sections. rice field.

2-5. Application to phosphorylated proteins other than phosphorylated tau For mice treated with normothermia (37°C) or hypothermia (< 30°C) (both males and females, aged 10-14 weeks), After anesthesia, the animal was fixed on a dissecting table and the abdomen was opened to expose the heart. Phosphate buffered saline was perfused from a peristaltic pump to an injection needle inserted into the left ventricle of the exposed heart to drain the blood.

After blood removal, the brain was quickly removed and freeze-frozen on a balance dish floated on liquid nitrogen. After confirming that it was completely frozen, it was stored at -80°C. Sections were prepared using a cryostat (Leica), sliced at a thickness of 15 μm, pasted on MAS-coated slide glasses, dried, and frozen at -80°C.

Place 3 pieces of Prowipe (Elleair) in a glass staining vat, absorb 30 mL of 4% paraformaldehyde, and preheat sufficiently on a hot plate set to 60°C. The fresh frozen sections prepared above were arranged here and fixed (fumigation fixation) at 60°C for 30 minutes. After fixation, they were washed with water for 5 minutes.

The tissue after fumigation was immersed in sodium dodecyl sulfate sample buffer for 5 minutes. Then, after washing with distilled water for 5 minutes, it was washed with TBS for 5 minutes.

Phosphor-GSK3β antibody (#9336) from Cell Signaling Technology was used as an antibody that recognizes phosphorylation of GSK3β. Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (#9101) from Cell Signaling Technology was used as an antibody that recognizes phosphorylation of ERK1/2. Tissues impregnated with sodium dodecyl sulfate sample buffer were stained with these antibodies.

Figure 7 is a diagram using the histological detection method of the present invention for other than phosphorylated tau, (A) Western blotting photograph of phosphorylated GSK3β, (B) Antibody staining of phosphorylated GSK3β. (C) Western blotting of phosphorylated ERK1/2, (D) Antibody staining of phosphorylated ERK1/2.

As shown in FIG. 7, both antibodies showed enhanced phosphorylation in the mouse brain under normothermic treatment and hypothermic treatment. This enhancement of phosphorylation cannot be detected by a normal tissue staining method, nor can a normal tissue staining method reveal a difference in staining between normothermic treatment and hypothermic treatment. On the other hand, according to the histological detection method of the present invention, #9336 showed staining in the cerebral cortex, hippocampus, striatum, etc., and in particular, extensive enhancement of staining in the gray matter of the brain. In #9101, signals were observed in mossy fibers in the CA3 region of the hippocampus, and hypothermia-induced enhancement was detected. From the above results, it is considered that the histological detection method of the present invention is useful not only for detecting phosphorylated tau but also for analyzing a wide range of phosphorylated proteins.

It can be used for research, drug development and diagnosis of Alzheimer's disease.

Claims (9)

a cryosectioning step of preparing a cryosection of a tissue containing a phosphorylated protein;
a fumigation step of treating the frozen section with water vapor of a tissue fixative to fumigate the tissue;
A pretreatment step of impregnating the fumigated tissue with an ionic surfactant before staining;
An antibody staining step of antibody staining the tissue impregnated with the ionic surfactant;
A method for histological detection of a phosphorylated protein, comprising: 2. The method for histological detection of phosphorylated protein according to claim 1, wherein said phosphorylated protein is phosphorylated tau. 3. The method for histological detection of a phosphorylated protein according to claim 2, wherein said phosphorylated tau is non-aggregated phosphorylated tau. 2. The method for histological detection of a phosphorylated protein according to claim 1, wherein said phosphorylated protein is phosphorylated GSK3β. 2. The method for histological detection of a phosphorylated protein according to claim 1, wherein said phosphorylated protein is phosphorylated ERK1/2. 6. The histological detection method of phosphorylated protein according to any one of claims 1 to 5, further comprising a blood removal step of perfusion blood removal before the frozen section preparation step. 7. The method for histological detection of phosphorylated proteins according to claim 1, wherein the tissue fixative is formalin. 8. The method for histological detection of phosphorylated proteins according to any one of claims 1 to 7, wherein the ionic surfactant is an anionic surfactant. a tissue fixative for treating cryosections of tissue containing phosphorylated proteins with steam to fumigate the tissue;
an ionic surfactant impregnated as a pretreatment into the fumigated tissue before antibody staining;
an antibody staining agent that stains the tissue impregnated with the ionic surfactant with an antibody;
A phosphorylated protein histological detection kit, characterized by comprising: