JP5070183B2 - Immunostaining method for lipid antigen in tissue - Google Patents

Description

  The present invention relates to a method for immunostaining lipid antigens in brain tissue. More particularly, the present invention relates to a method for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells using cholesterol-specific surfactants, compositions and kits therefor.

  Oligodendrocytes (OL) are responsible for myelin formation in the central nervous system. The differentiation of OL lineage cells, including OL progenitor cells (OPCs), premyelinating OLs and myelinating OLs, is characterized by OL lineage specific glycolipids and proteins that are expressed sequentially. Immunohistochemical detection of these antigens allows identification of the differentiation stage of OL lineage cells during development.

  The most abundant myelin lipid is galactocerebroside (GalC) and its sulfated derivative, sulfatide (SUL), which together account for about 27% of the myelin lipid. This abundance and cell specificity makes GalC / SUL particularly useful as one of the earliest markers of OL that has terminated cell division. Furthermore, since GalC / SUL immunoreactivity is distributed almost throughout the OL cell membrane, GalC / SUL is useful for visualizing OL morphological changes during development.

  Furthermore, in mice deficient in the GalC / SUL biosynthetic enzyme UDP-galactose ceramide galactosyltransferase (CGT), the OL protrusions are not properly located along the axons, resulting in defects in nerve conduction and Lambie restriction. Abnormal formation of the ring occurs.

  Involvement of GalC and anti-GalC antibodies in pathological conditions has been reported. It has been shown that Krabbe disease (also known as globuloid cell white matter dystrophy) is caused by dysfunction of the GalC catalytic enzyme galactocerebrosidase activity. In Krabbe disease, GalC has been reported to accumulate in vivo. GalC is also involved in demyelinating diseases. Anti-GalC antibodies have been detected in patients with post-infection encephalitis and Guillain-Barre syndrome. Furthermore, anti-GalC antibodies have been reported to cause experimental allergic neuritis. The possible role of GalC in the entry of human immunodeficiency virus (HIV) into neurons and mucosal epithelium has also been reported.

  The above results suggest that analysis of GalC / SUL expression in vivo is critical for understanding OL differentiation and myelin function.

  The expression level and distribution of GalC / SUL in cultured OL has been extensively studied. However, it has been reported that the GalC expression level in Schwann cells changes rapidly with time during the culture, suggesting that the GalC expression level in the cultured cells may be different from the in vivo expression level. . It is therefore desirable to examine the expression level and distribution pattern of GalC in tissue sections. However, the expression of GalC / SUL in vivo has not been studied extensively. This is because analysis of the immunohistochemical localization of GalC / SUL in tissue sections is not easy.

  As with other lipid molecules and lipophilic tracers, the GalC / SUL immunoreactivity is triton X-, a surfactant commonly used to enhance antibody penetration into tissue sections. 100 (Non-patent Documents 1 and 2). This is presumably because GalC / SUL was eluted from the cell membrane of OL by Triton X-100.

  There is a possibility that this problem can be solved by omitting the permeabilization step in the immunostaining protocol Triton X-100. In fact, the distribution pattern of GalC in a fixed brain slice has been examined without permeation processing (Non-Patent Documents 1 to 4). However, when a fixed tissue section was immunostained without permeabilization, only the antigen located on the surface of the section was labeled, and the antigen located in the center of the section remained unstained (Non-patent Document 5). Therefore, it was difficult to comprehensively examine the three-dimensional distribution of GalC / SUL, morphological differentiation and OL number in the section without using a surfactant. Warrington et al. Clearly visualized the distribution pattern of glycolipids in tissue sections using unfixed tissue sections (Non-patent Document 6). However, since anti-glycolipid antibodies often exhibit biological activity against cultured OL, incubation of unfixed tissue with anti-glycolipid antibodies may have an unexpected effect on OL.

  Methanol and ethanol may also be used to enhance antibody penetration into brain sections. Although these reagents effectively improve the penetration of antibodies into tissue sections, they solubilize a wide range of lipids almost indiscriminately (7), so these reagents were used for immunostaining. In some cases, detection of lipid antigens is often difficult.

Therefore, it was desired to establish a new immunohistochemical method that enhances the penetration of antibodies into tissue sections without inhibiting the GalC / SUL expression distribution.
Ghandour MS et al., J Neurocytol, 1988; 17: 485-98 Reynolds R et al., Development, 1988; 102: 409-25 Curtis R et al., J Neurocytol, 1988; 17: 43-54 Hardy R et al., Development, 1991; 111: 1061-80 Matsubayashi Y et al., J Neurosci Methods, 2008; 174: 71-81 Warrington AE et al., J Neurosci Res, 1992; 33: 338-53 Schuck S etal., Proc Natl Acad Sci USA, 2003; 100: 5795-800

  We set out to create a new immunohistochemical procedure for visualizing GalC / SUL using fixed tissue sections. Recently, the inventors have developed an immunohistochemical method for tissue sections labeled with DiI, a lipophilic neuronal tracer (Non-Patent Document 5). Instead of Triton X-100, which solubilizes lipid molecules almost indiscriminately (Non-Patent Document 7), the present inventors permeabilized DiI-labeled sections using digitonin, which is a cholesterol-specific surfactant. The present inventors have found that treatment of DiI labeled brain sections with digitonin can efficiently penetrate the antibody into the tissue without destroying the DiI label in the neurons.

  Similarly, the present inventors thought that digitonin does not inhibit the distribution pattern of GalC / SUL, which is an endogenous glycolipid. Consistent with this hypothesis, we show here that digitonin treatment allows the antibody to penetrate efficiently into tissue sections without interfering with GalC / SUL labeling. Digitonin is often used in cell biological and biochemical assays such as membrane permeabilization and protein extraction, but to the best of our knowledge, immunofluorescence of endogenous glycolipid antigens in brain sections. This is the first time digitonin has been applied for staining. Visualization of the GalC / SUL distribution in fixed brain sections is useful for a detailed study of GalC / SUL expression in both physiological and pathological conditions.

  Accordingly, an object of the present invention is to provide a method for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells.

The present invention
[1] A step of contacting an oligodendrocyte lineage cell with a cholesterol-specific surfactant, and an oligodendrocyte lineage cell contacted with a cholesterol-specific surfactant and an antibody specific for galactocerebroside and / or sulfatide A method for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells, comprising the step of:
[2] The method of [1], wherein the cholesterol-specific surfactant is digitonin.
[3] A composition for promoting penetration of an antibody specific for galactocerebroside and / or sulfatide into oligodendrocyte lineage cells, comprising a cholesterol-specific surfactant;
[4] The composition of [3], wherein the cholesterol-specific surfactant is digitonin;
[5] A kit for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells, comprising a cholesterol-specific surfactant and an antibody specific for galactocerebroside and / or sulfatide;
[6] The kit according to [5], wherein the cholesterol-specific surfactant is digitonin.
About.

  The present invention provides a method for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells using cholesterol-specific surfactants, compositions and kits therefor.

  “Oligodendrocyte lineage cells” include, in addition to oligodendrocytes (OL), cells such as oligodendrocyte progenitor cells (OPC) generated in the course of oligodendrocyte development. In the central nervous system, myelin is formed from the process of oligodendrocytes, but both pre-myelinating oligodendrocytes that are not myelinating and oligodendrocytes that are myelinating are oligodendrocyte lineage cells. included.

  “Galactocerebroside (GalC)” is a glycosphingolipid in which galactose is β-glycoside-bonded to the primary alcoholic hydroxyl group of ceramide, and is a constituent glycolipid of brain myelin. “Sulphatide (SUL)” is a galactocerebroside galactose in which a sulfate group is ester-bonded to the C-3 position, and is also a constituent glycolipid of brain myelin. These are the earliest markers of mitotic oligodendrocytes. Although the present invention is described mainly for visualization of galactocerebroside and / or sulfatide in oligodendrocyte lineage cells, galactocerebroside is also expressed in cells other than oligodendrocyte lineage, such as the epithelium of the digestive system. In addition, since substantially all membranes can be permeabilized by the present invention as described below, the present invention is applied to any cell in which these glycolipids may be expressed. be able to.

As used herein, the term “an antibody specific for galactocerebroside and / or sulfatide” refers to an antibody that specifically recognizes one or both of galactocerebroside and sulfatide. The antibody may be a polyclonal antibody, but a monoclonal antibody is more preferable for obtaining a homogeneous result. An antibody also encompasses any form (eg, Fab, F (ab ′) ₂ , scFv, etc.) having the ability to specifically bind to an antigen. Examples of such antibodies include the monoclonal antibody R-mAb that recognizes both galactocerebroside and sulfatide and other structurally related trace lipids (Ranscht B et al., Proc Natl Acad Sci USA, 1982). 79: 2709-13; Bansal R et al., J Neurosci Res, 1989; 24: 548-57).

  Any known label can be used to visualize the antibody bound to the antigen. Examples of preferred labels include fluorescent labels. The antibody may be labeled directly or indirectly using a labeled secondary antibody or the like.

  According to the present invention, multiple staining of brain tissue sections can be performed by using an antibody against another antigen in addition to an antibody specific for galactocerebroside and / or sulfatide. The present inventors have shown that nuclear antigen immunostaining is possible even when digitonin, which is a cholesterol-specific surfactant, is allowed to act on a nuclear membrane having a low cholesterol content. Accordingly, an antibody against an antigen in the nucleus can be used as another antibody. In such a case, the respective antigens can be visualized simultaneously by labeling each antibody with fluorescence having a different wavelength.

  It is known that cell membranes contain lipids such as phospholipids, glycolipids, and steroids (eg, cholesterol). These are classified into glycerolipids whose alcohol component in the hydrophobic part is glycerol and sphingolipids whose component is sphingosine. As used herein, the term “cholesterol-specific surfactant” refers to a surfactant that specifically acts on cholesterol among the lipids in the cell membrane. As the cholesterol-specific surfactant, any known saponin such as digitonin and kiraya saponin can be used. Saponin is a general term for glycosides having steroids and triterpenoids as non-sugar parts, and digitonin, a steroid glycoside derived from digitalis, is also included in saponins. Derivatives of these cholesterol specific surfactants can be used in the present invention as well. Thus, as used herein, the term “digitonin” includes any derivative thereof. Digitonin is preferably used in the present invention. Hereinafter, the present invention will be mainly described with respect to digitonin.

  Surfactant is a general term for substances having properties that change the properties of the interface or surface (surface activity), such as a reduction in the surface tension of water, and its actions are diverse, but for the purposes of the present invention, Those having the property of acting to break down lipids are preferably used. The “action” of the surfactant on the lipid is not limited in its mechanism as long as the penetration of the protein specifically binding to the target substance into the tissue is promoted. For example, digitonin precipitates with cholesterol, forming an insoluble complex called cholesterol-digitonin.

  The concentration of the cholesterol-specific surfactant at the time of contact with the oligodendrocyte lineage cell can vary depending on the brain tissue used and the like, and is appropriately determined based on the result of immunostaining. For example, in the case of digitonin, the concentration is, for example, 10 μg / ml or more, preferably 30 μg / ml or more, more preferably 60 μg / ml or more, and for example 500 μg / ml or less, preferably 250 μg / ml or less, more preferably 120 μg / ml or less. is there.

  In the present invention, brain tissue sections that may contain oligodendrocyte lineage cells can be used for visualization of oligodendrocyte lineage cells. The brain tissue section can be prepared using a known means such as a vibratome.

  The present invention further provides compositions and kits for use in the above methods. The composition of the present invention comprises a cholesterol-specific surfactant as described above. The kit of the present invention also comprises a cholesterol-specific surfactant as described above and an antibody specific for galactocerebroside and / or sulfatide as described above. These compositions and kits may contain any additional components (eg, antibodies to another antigen, visualization reagents, buffers, preservatives, etc.).

  By performing in vivo immunohistochemical localization of galactocerebroside and / or sulfatide according to the present invention, oligodendrocyte development, differentiation, morphological changes, myelin formation, function, etc. can be studied. Further, by visualizing galactocerebroside and / or sulfatide according to the present invention, such as Krabbe disease (globoid cell leukodystrophy), demyelinating disease, post-infection encephalitis, Guillain-Barre syndrome, allergic neuritis, AIDS, etc., It is possible to study the function of these glycolipids in diseases in which the involvement of galactocerebroside and / or sulfatide is suggested, and to diagnose diseases.

Materials and Methods Animals: ICR and C57BL / 6J mice were purchased from Japan SLC. Olig1-deficient mice (Xin M et al., J Neurosci, 2005; 25: 1354-65) were maintained in a hybrid background of 129 and C57BL / 6 and the genotypes of Olig1-deficient mice were determined as described. (Toda T et al., Mol Cell Neurosci, 2008; 39: 40-9). All mice were housed on a regular 12 hour light / dark schedule.

  Antibodies and reagents: Anti-GalC / SUL antibody ("R-mAb") was obtained from Dr. Barbara Ranscht (the Burnham Institute, La Jolla, CA) (Ranscht B et al., Proc Natl Acad Sci USA, 1982; 79: 2709-13). O4, NeuN, Cy3-conjugated anti-rabbit IgG and Cy3-conjugated anti-mouse IgG and IgM antibodies were obtained from Chemicon. Anti-myelin basic protein (MBP) antibody and CC1 antibody were obtained from Covance and Calbiochem, respectively. Alexa488 conjugated anti-mouse IgG3 and Alexa568 conjugated anti-mouse IgG1 antibodies were obtained from Molecular Probes. Cy3-conjugated anti-mouse IgG2 antibody was obtained from Jackson ImmunoResearch. Digitonin was purchased from Calbiochem and stocked at −20 ° C. at a concentration of 50 mg / ml in dimethyl sulfoxide (DMSO, Sigma). In use, the stock solution was diluted at least 100-fold.

  Brain section preparation: Mice were deeply anesthetized and perfused transcardially with phosphate buffered saline (PBS) containing 4% paraformaldehyde (PFA) (4% PFA / PBS). The brain was removed and postfixed with 4% PFA / PBS overnight. The brains were then sectioned using a vibratome (thickness 30 μm) and stored in PBS containing 0.03% sodium azide.

  Immunohistochemistry: Immunohistochemistry was performed by modifying a previously described method (Kawasaki, H., et al., Neuron, 2000; 28: 31-40; Non-Patent Document 5). Brain sections were pretreated with DMSO (0.24% -1%), digitonin (60-500 μg / ml) or 3% bovine serum albumin (BSA) / PBS containing 0.5% Triton X-100 for 30 minutes at room temperature. Treated and incubated with primary antibody in each pretreatment solution for 12 hours at 4 ° C. For permeabilization with methanol, sections were incubated in methanol for 30 minutes at −20 ° C., washed with PBS, and then incubated with primary antibody in 3% BSA / PBS for 12 hours at 4 ° C. Following incubation with primary antibody, sections were washed with PBS, incubated with secondary antibody and Hoechst 33342 (final concentration 1 μg / ml, purchased from Calbiochem) in 3% BSA / PBS for 3 hours at room temperature and washed with PBS . The sections were then attached to a microscope slide and a cover glass was placed using Mowiol (Calbiochem). Antibody dilutions were as follows: anti-GalC / SUL, 1: 100; CC1, 1: 1000; anti-MBP, 1: 1000; O4, 1: 500; anti-NeuV, 1: 1000; Cy3-conjugated anti-mouse IgG2, 1: 1000; other secondary antibodies, 1: 500.

  Microscopic observation: Microscopic analysis was performed as previously described (Non-Patent Document 5). Epi-illumination microscopy was performed using an AxioImager A1 microscope (Carl Zeiss). Confocal microscopy was performed using an LSM510 microscope and a 40x / 1.3 NA objective (Carl Zeiss). Z stack images (optical thickness 2.1 μm) were collected every 1.75 μm and reconstructed three-dimensionally (3D) using ZEN 2007 and ZEN 2007 LE software (Carl Zeiss). The image was shown as a single XY or XZ optical cross section or as an XZ maximum projected image, where a 3D view of the data was calculated and displayed by showing only the highest intensity pixels along the projection axis. In order to quantify the immunofluorescence intensity across the section, a rectangular basin of interest (ROI) was drawn in the XZ maximum projection image across the entire width (220 μm) and depth (approximately 20 μm) of the brain section. Horizontal average pixel intensity was measured and plotted against depth using ImageJ (public domain software, http://rsb.info.nih.gov/ij/).

  Data analysis of NeuN immunoreactivity subcellular localization: Quantitative analysis of NeuN immunoreactivity subcellular localization was performed by modifying a previously described method (Matsubayashi Y et al., J Biol Chem, 2001). ; 276: 41755-60). Intracellular localization of NeuN immunoreactivity in the middle of each tissue section was visually examined and classified into two categories: “N ≧ C”, NeuN nuclear immunoreactivity is stronger than cytoplasmic immunoreactivity “N <C”, cytoplasmic immunoreactivity is stronger than nuclear immunoreactivity. The number of cells classified into each category was counted and used for further statistical analysis. To assess statistical significance, the absolute number of cells classified into each category is listed in a 2 × 2 contingency table (eg, DMSO treated samples versus digitonin treated samples and “N ≧ C” vs. “N <C”. ), And analyzed by the Mantel-Henzel procedure using Statcel 2 software (OMS Publishing).

Example 1 Effects of Digitonin and Other Permeation Reagents on GalC / SUL Immunoreactivity in Brain Sections First, the effects of various permeation reagents on GalC / SUL immunoreactivity in mouse brain sections were compared. Recognizes both GalC and SUL as well as other structurally related lipids (Bansal R et al., J Neurosci Res, 1989; 24: 548-57) Monoclonal antibody R-mAb (Ranscht B et al., Proc Natl Acad Sci USA, 1982; 79: 2709-13). Hereinafter, this antibody is referred to as “anti-Gal / SUL antibody”. The fixed brain of the P8 mouse was removed and a coronal section was prepared using a vibratome. The sections were then double stained with anti-GalC / SUL and CC1 antibodies using control 3% BSA / PBS, 0.4% DMSO, 120 μg / ml digitonin, 0.5% Triton X-100 or methanol. . The CC1 antibody recognizes APC, a protein that is prominently expressed in the OL cell body (Bhat RV et al., Glia, 1996; 17: 169-74). Since it was found that the immunoreactivity of CC1 was not significantly inhibited by various permeation reagents, the CC1 antibody was used to visualize the location of OL cell bodies. When sections were stained using BSA / PBS alone, GalC / SUL and CC1 immunoreactivity was detected only on the surface of the sections. There were often GalC / SUL positive spots around GalC / SUL and CC1 positive OL cell bodies. These points appear to indicate OL protrusions on the surface of the section. Substantially similar results were obtained when the sections were treated with DMSO, a solvent for digitonin. In contrast, in sections treated with 120 μg / ml digitonin, GalC / SUL positive processes were stained more clearly and continuously than those treated with BSA / PBS or DMSO. This result suggests that digitonin treatment enhances the penetration of the antibody into the section and at the same time does not inhibit the distribution of GalC / SUL. When sections were treated with Triton X-100, GalC / SUL immunoreactivity was disrupted and numerous bleb-like labels were seen around CC1-positive OL cell bodies. Also, GalC / SUL immunoreactivity disappeared almost completely after methanol treatment. These results are consistent with previous reports that Triton X-100 and organic solvents are not compatible with immunostaining of GalC / SUL and other lipid molecules (Non-Patent Document 2; Schwarz A et al., Methods Enzymol, 2000). 312: 179-87). Therefore, digitonin was used in the experiment below.

Example 2: Analysis of GalC / SUL positive structure in digitonin-treated sections The immunostaining method of the present invention using digitonin allowed examination of the colocalization of GalC / SUL and other OL markers with high spatial resolution. Therefore, the characteristics of the GalC / SUL positive structure in the cerebral cortex were analyzed next. In digitonin (60 μg / ml) treated sections, two morphologically different OLs were labeled with anti-GalC / SUL antibody. The first OL had complex rosette-like projections extending radially from the cell body. Double staining showed that most of these OLs were CC1 positive and O4 positive. The second OL occasionally had a few short simple protrusions. Double staining indicated that these cells were CC1 negative and O4 positive. Because of its simple form, the latter is considered immature than the former. In addition to these structures, a relatively linear fibrous structure, most of which extends from white matter to cortex, was labeled with anti-GalC / SUL antibody. Double immunostaining showed that this GalC / SUL positive fibrous structure was also positive for MBP. This suggests that these fibrous structures are myelin sheaths. These results suggest that OL is clearly labeled with anti-GalC / SUL antibody in digitonin-treated sections.

  In order to eliminate the possibility that digitonin treatment changes the nature of antigens present in the cerebral cortex, so that anti-GalC / SUL antibodies may recognize unexpected antigens present in cells other than OL after digitonin treatment. Whether or not the GalC / SUL immunoreactivity disappeared in the absence of OL was examined using Olig1-deficient mice. Olig1 is a basic helix-loop-helix transcription factor essential for OL differentiation (Lu QR et al., Cell, 2002; 109: 75-86; Zhou Q et al., Cell, 2002; 109: 61- 73), OL is markedly reduced in Olig1-deficient mice (Xin M et al., J Neurosci, 2005; 25: 1354-65). When brain sections derived from Olig1-deficient mice were stained with anti-GalC / SUL antibody using digitonin (120 μg / ml), the GalC / SUL immunoreactivity was significantly reduced. Taken together, the above results strongly suggest that GalC / SUL can be reliably detected with anti-GalC / SUL antibody using digitonin treatment.

Example 3: Concentration-dependent effect of digitonin on GalC / SUL immunoreactivity To find the optimum concentration of digitonin, the effect of various concentrations (60-500 μg / ml) of digitonin on GalC / SUL immunoreactivity was examined. did. P9 mouse brain sections were stained with anti-GalC / SUL antibody using various concentrations of digitonin. In sections treated with 60 μg / ml digitonin, OL protrusions and myelin were labeled more clearly and continuously than sections treated with control 3% BSA / PBS or 1% DMSO. OL and myelin were also clearly detected in sections treated with higher concentrations (120-500 μg / ml) of digitonin, but probably due to some degradation of GalC / SUL immunoreactivity. A bull-like structure appeared depending on the digitonin concentration. GalC / SUL immunoreactivity in O4 ⁺ CC1 ⁻ cells disappeared depending on the digitonin concentration, and could not be detected when the sections were treated with digitonin at a concentration of 250 μg / ml or more. These results suggest that the optimal concentration of digitonin is about 60-120 μg / ml. GalC / SUL immunoreactivity on O4 ⁺ CC1 ⁺ OL and MBP ⁺ myelin was relatively stable even in the presence of high concentrations (250-500 μg / ml) of digitonin. The reason for the difference in sensitivity to digitonin from O4 ⁺ CC1 ⁻ cells is expected to be different in R-mAb antigens on these cells and different in sensitivity to digitonin, but is unknown at this time. In any case, since the transmission efficiency varies depending on the tissue, it is practically preferable to determine an appropriate digitonin concentration according to the cell type and structure to be visualized.

  Next, it was examined how deeply the anti-GalC / SUL antibody penetrates into a digitonin-treated cerebral cortex slice. P9 mouse brain sections were stained with anti-GalC / SUL antibody using 3% BSA / PBS, 1% DMSO or 60-500 μg / ml digitonin, and then shared with GalC / SUL immunoreactivity in cerebral cortex layer VI Considered by using focal microscopy. In control BSA / PBS and DMSO treated tissues, the central immunoreactivity of the sections was faint. Consistent with this, when the immunofluorescence intensity was measured and plotted against the depth of the section, the intensity profile showed a deep valley in the middle of the section. In contrast, in the digitonin-treated sample, the immunoreactivity in the middle of the section was significantly enhanced depending on the digitonin concentration. Thus, the valleys of the intensity-depth plot were significantly shallower in the digitonin-treated sections. These results demonstrate that digitonin treatment significantly enhances antibody penetration into cerebral cortex tissue sections. Combined with our previous report that digitonin also enhances antibody penetration into cerebellar slices (Non-Patent Document 5), these results indicate that digitonin treatment can be applied to various brain regions. is doing.

  As described above, GalC / SUL-labeled bleb-like structures appeared depending on the digitonin concentration. This suggests that digitonin treatment can cause some degradation of GalC / SUL immunoreactivity. Importantly, only a small number of blebs appeared when sectioned with 60 μg / ml digitonin. Furthermore, even when the sections were treated with higher concentrations of digitonin, confocal microscopy showed that these blebs were confined to the surface of the sections, and digitonin treatment reduced the GalC / SUL immunoreactivity in the middle of the sections. A significant improvement was demonstrated. Taken together, these results demonstrate that digitonin treatment significantly enhances GalC / SUL immunoreactivity with minimal degradation, especially when sections are treated with 60-120 μg / ml digitonin. In fact, 120 μg / ml digitonin, not DMSO, was used to successfully visualize OL protrusions throughout the depth of the section.

Example 4: Nuclear membrane permeabilization using the method of the present invention Cholesterol, the target of digitonin, is unevenly distributed in the cell membrane. One membrane that contains the least amount of cholesterol and is therefore most resistant to digitonin is the nuclear membrane (Elias PM et al., J Cell Biol, 1978; 78: 577-96; Adam SA et al., J Cell Biol, 1990; 111: 807-16; van Meer G et al., Nat Rev Mol Cell Biol, 2008; 9: 112-24). Therefore, the method of the present invention may have a limitation that it cannot be applied to immunostaining of nuclear antigens. If so, double staining with anti-GalC / SUL antibody and other antibodies against nuclear antigens is not possible. However, the inventors have previously demonstrated that even nuclear antigen can be successfully detected using digitonin at a concentration of 1000 μg / ml (Non-Patent Document 5). Since 1000 μg / ml digitonin degraded GalC / SUL immunoreactivity, it was examined here whether lower concentrations of digitonin are sufficient for nuclear membrane permeabilization and detection of nuclear antigens. Cortical sections are expressed in various concentrations (60-100 μg / ml) of digitonin with antibodies against NeuN, a protein that is expressed in most neurons of the mouse brain and is present in the cell nucleus and cytoplasm. (Mullen RJ et al., Development, 1992; 116: 201-11; Lind D et al., J Neurosci Res, 2005; 79: 295-302). The NeuN immunoreactivity in the middle of the sections was then analyzed using confocal microscopy.

  In control 1% DMSO treated sections, NeuN immunoreactivity was weak in the middle of the section. Furthermore, this weak NeuN immunoreactivity was localized mainly in the cytoplasm and less frequently in the nucleus. The percentage of neurons with NeuN immunoreactivity in the nucleus equal to or higher than NeuN immunoreactivity in the cytoplasm (hereinafter this ratio is referred to as “N ≧ C” value) was about 55%. In these cells, it is likely that nuclear NeuN was labeled with the antibody due to accidental physical disruption of the nuclear envelope that occurred during vibratome sectioning. Substantially similar results were obtained for 3% BSA / PBS treated sections.

  In contrast, in digitonin-treated sections, the “N ≧ C” value increased significantly depending on the digitonin concentration and reached a plateau value of about 93% when the digitonin concentration was increased to 120 μg / ml or higher. . Thus, in the middle of brain sections treated with 120 μg / ml digitonin, nuclear NeuN immunoreactivity was successfully detected in almost all NeuN positive cells. Taken together, these results demonstrate that even antigens in the cell nucleus can be successfully detected using the method of the present invention by using digitonin at a concentration of 120 μg / ml. Indeed, using this digitonin concentration, double staining with anti-GalC / SUL and anti-NeuN antibodies was also possible throughout the depth of the tissue section.

  As explained above, since the nuclear membrane is considered to be the most resistant membrane to digitonin, these results indicate that substantially all membranes can be sufficiently permeabilized with 120 μg / ml digitonin. I can guess. Thus, increasing digitonin concentrations above 120 μg / ml has little additional effect on membrane permeability and may increase the risk of degradation of GalC / SUL immunoreactivity.

Example 5: Effect of digitonin on O4 immunoreactivity To investigate whether the method of the present invention can be applied not only to GalC / SUL but also to other antigens that are vulnerable to permeation reagents, against O4 immunoreactivity. The effect of digitonin was examined. The O4 antibody recognizes multiple antigens expressed by OL and OPC, including SUL and the unidentified molecule POA (Sommer I et al., Dev Biol, 1981; 83: 311-27; Bansal R et al , J Neurosci Res, 1989; 24: 548-57; Bansal R et al., J Neurochem, 1992; 58: 2221-9). Importantly, O4 immunoreactivity is inhibited by acetone (Sommer I et al., Dev Biol, 1981; 83: 311-27) or Triton X-100 (unpublished data). P9 mouse brain sections were stained with O4 antibody using BSA / PBS, DMSO or 60-250 μg / ml digitonin. O4 immunoreactivity was clearly detected on the surface of BSA / PBS, DMSO-treated sections, but was significantly reduced even at a concentration of 60 μg / ml in digitonin-treated sections. When the sections were treated with higher concentrations (120 and 250 μg / ml) of digitonin, the immunoreactivity disappeared almost completely. Therefore, O4 immunoreactivity may be more sensitive to digitonin than GalC / SUL immunoreactivity. Digitonin at concentrations below 60 μg / ml may be compatible with O4 immunostaining, but this is not discussed. This is because more than 60 μg / ml digitonin is required for efficient detection of nuclear NeuN immunoreactivity in the middle of the section. These results suggest that the sensitivity to digitonin varies between the various lipid antigens and not all types of lipid molecules can be tested using digitonin.

  Recent studies have shown that digitonin treatment maintains the intracellular localization of the endogenous lipid antigen phosphatidylinositol 4,5-bisphosphate (PI (4,5) P2) in HL-60 cells. (Sharma VP et al., J Leukoc Biol, 2008; 84: 440-7). Moreover, the present inventors have shown that digitonin treatment is compatible with DiI which is a lipophilic neuron tracer (Non-patent Document 5). These findings and the detection of the glycolipid antigen GalC / SUL herein may suggest the possibility that digitonin treatment can be used to examine the localization of other lipids or lipophilic tracers by immunostaining. . However, as shown for O4 in this example, not all lipids can be visualized using digitonin. Finding a law for predicting whether digitonin treatment is useful for preserving the localization of a specific lipid molecule based on the structure of the molecule is a future task.

  The present invention provides a method for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells using cholesterol-specific surfactants, compositions and kits therefor.

Claims (3)

Contacting oligodendrocyte lineage cells with a cholesterol specific surfactant, and contacting oligodendrocyte lineage cells contacted with a cholesterol specific surfactant with an antibody specific for galactocerebroside and / or sulfatide. A method for visualizing galactocerebroside and / or sulfatide in oligodendrocyte lineage cells, comprising the step , wherein the cholesterol-specific surfactant is digitonin . A composition for promoting penetration of an antibody specific for galactocerebroside and / or sulfatide into oligodendrocyte lineage cells, comprising a cholesterol specific surfactant, wherein the cholesterol specific surfactant is digitonin, Composition . A kit for visualization of galactocerebroside and / or sulfatide in oligodendrocyte lineage cells comprising a cholesterol specific surfactant and an antibody specific for galactocerebroside and / or sulfatide , wherein the cholesterol specific surfactant is A kit that is digitonin .