Method 1: The sections are dried overnight in a 60 oven. Slides are then deparaffinized and rehydrated to distilled water in three changes of xylene, five minutes each, two changes of 100% alcohol, three minutes each, and one change of 95% alcohol for three minutes, then rinsed well in distilled water. Following this, sections are treated for thirty minutes with 98% formic acid followed by washing and then holding over boiling distilled water in a closed container for 30 minutes. After this, the slides are allowed to cool. The slides are then incubated for five minutes in Tris-buffered saline (TBS) containing 0.05% Tween 20 (TBST) and placed in an autostainer (Dako Cytomation). All subsequent steps are performed on the autostainer withTBST buffer rinse steps between each reagent. Immunostaining reagents are obtained in a kit from the supplier (Envision+System-HRP-DAB, Dako Cytomation). Endogenous peroxidase is first quenched with 0.03% hydrogen peroxide for five minutes. This is followed by incubations in normal goat serum (1:20) for twenty minutes, then rabbit polyclonal primary antiserum, NACP 98 (14) at 1:3,000 for two hours, anti-rabbit Labeled Polymer-HRP for thirty minutes, distilled water rinse for three minutes and 3,3’-diaminobenzidine, supplied premixed with buffer containing hydrogen peroxide. Counterstaining with hematoxylin, dehydration and mounting steps are then performed manually.
Method 2: Sections are deparaffinized in five changes of xylene, ten minutes each, then rehydrated in three changes of absolute alcohol, three changes of 95% alcohol and deionized water. Sections are then incubated, for epitope retrieval, in a working solution of proteinase K (Enzo Life Sciences) diluted in phosphate buffer (Dako Cytomation) in a waterbath at 37 C for twenty minutes followed by a ten minute cooldown and deionized water rinse. The appropriate dilution of proteinase K is determined by performing the full staining protocol with a series of dilutions on positive control sections; the range for this is 1:100 – 1:800. Further epitope retrieval is then performed using Reveal™ heat retrieval solution (Biocare Medical) in a waterbath at 98 degrees C for fifteen minutes followed by a ten minute cooldown and deionized water rinse, after which slides are transferred to phosphate buffered saline (PBS). Staining is performed on a Benchmark XT automated immunostainer with proprietary reagents (Ventana Medical Systems). Sections are incubated for thirty-two minutes in mouse monoclonal anti-α-synuclein primary antibody (LB509, Invitrogen) at a 1:80 dilution in ChemMate diluent. Bound primary antibody is then localized using the ultraVue™ Universal Alkaline Phosphatase Red Detection Kit, which is a biotin-free, multimeric compound consisting of a mixture of enzyme labeled secondary antibodies that are then visualized with naphthol and Fast Red chromogen. Sections are then counterstained with hematoxylin, dehydrated, coverslipped with permanent mounting medium, and evaluated by light microscopy.
Method 3: Six mm thick paraffin sections are placed on X-tra Slides, (Surgipath) and dried in an oven overnight at 58C. Following removal from the oven, slides are immediately deparaffinized in xylene and brought through graded alcohols to distilled water. The slides are then incubated for 5 minutes at 37C in protease solution that consisted of 300 ml distilled water with 100 mg of type XXIV Protease (Sigma). The sections are washed gently in running water, put in PBS (Scytek) for 10 minutes, after which Protein Blocking Agent (ThermoFisher) is applied for 20 minutes. Excess reagent is then blotted off and slides are incubated for 60 minutes at room temperature with primary antibody (1:100, clone LB509, Zymed) diluted in Common Antibody Diluent (Biogenex). After being rinsed in PBS, the slides are incubated for 30 minutes with biotinylated anti-mouse IgG (LSAB-2 kit, Dako). Following a 10 minute PBS wash, sections are incubated 30 minutes with streptavidin (Dako LSAB-2 kit), rinsed in PBS, and developed with freshly prepared and filtered DAB (Sigma) and hydrogen peroxide. Sections are then counterstained with Mayer’s hematoxylin (Sigma).
Method 4: Sections are deparaffinized in xylene and rehydrated in a graded series of alcohols to water. After blocking with 10% calf serum in TBS (CTBS), sections are incubated in the same solution with a 1:2000 dilution of the monoclonal antibody, PSyn#64 (Wako), raised against -synuclein phosphorylated at serine 129, at room temperature overnight. After washing in TBS, sections are then treated with biotinylated anti-mouse IgG diluted in CTBS for 2 hours at room temperature. After rinsing in TBS, sections are treated with ABC (Vector Laboratories), rinsed again in TBS and then developed with DAB and hydrogen peroxide for 5 minutes and counterstained with hematoxylin.
Method 5: Paraffin sections are dried in an oven at 37C overnight. Sections are deparaffinized in three changes of xylene, ten minutes each, and two changes of 100% ethanol, 10 minutes each.Endogenous peroxidase is then suppressed with 1% hydrogen peroxide dissolved in methanol for thirty minutes. Sections are then rehydrated in 90% and 70% ethanol followed by distilled water, five minutes each. Sections are then treated with 100% formic acid for five minutes, rinsed in water for five minutes and then two changes of PBS for five minutes. Non-specific staining is then blocked with 10% horse serum (Vector Laboratories) in PBS for 20 minutes. Primary antibody is then applied (BD Transduction Labs, catalogue # 610787) at 1:300 dilution in PBS and the slides incubated overnight at four degrees C. After four washes in PBS, five minutes each, slides are treated with biotinylated horse anti-mouse IgG (Vector Laboratories) preadsorbed with 1:100 heat-inactivated normal human serum (Scipac)diluted at 1:100 in PBS, for one hour at room temperature. After washing again in PBS, sections are incubated with ABC (Vector Laboratories) for one hour at room temperature, washed again in PBS and then developed for three minutes with 0.25mg/ml DAB in PBS with four drops/100ml of 30% hydrogen peroxide. The slides are then counterstained with hematoxylin.
Method 6: Sections are deparaffinized in xylene, rehydrated in graded ethanol washes and immersed in a fresh methanol/30% hydrogen peroxide bath for thirty minutes to quench endogenous peroxidase activity. After several washes in water, sections are transferred to boats filled with R-Buffer U(Electron Microscopy Sciences) and processed overnight in the Antigen Retriever 2100 (Electron Microscopy Sciences). The following day, sections are removed from the Antigen Retriever and washed with water before immersion in 88% formic acid for thirty minutes.Slides are then washed in water and transferred to 0.1M Tris buffer, followed by 0.1M Tris buffer/2% dilute goat serum for blocking of non-specific binding. Slides are incubated overnight at 4°C in Syn303 monoclonal antibody (17) at 1:16,000 dilution in 0.1M Tris buffer/2% dilute goat serum. Following washes in Tris buffer, slides are incubated in biotinylated anti-mouse IgG at 1:1000 dilution for one hour at room temperature. Following rinsing of the secondary antibody, slides are processed with ABC (Vector Laboratories) for one hour, and then developed DAB with imidazole and hydrogen peroxide.
Method 7: Sections are deparaffinized in xylene and hydrated through graded alcohols to distilled water. Sections are then pretreated with 88% formic acid for 5 minutes, rinsed in PBS, and then with 3% hydrogen peroxide. After a 60-minute block in 5% milk powder, sections are incubated at room temperature with the Syn 303 monoclonal antibody (21,17)for one hour. After a PBS rinse the sections are incubated with secondary antibody biotinylated anti-mouse IgG for 45 minutes. After an additional PBS rinse, the sections are incubated in ABC for one hour (Vector Laboratories). Sections are incubated in DAB with hydrogen peroxide for ten minutes, rinsed, and then dipped in lithium carbonate solution, rinsed with tap water, dehydrated through graded alcohols, and cleared in xylene.
Method 8: Paraffin-embedded sections are dried in the oven for one hour and then hydrated though three changes of xylene, two changes of 100% alcohol and then 95%, 75% alcohol and distilled water. Endogenous peroxidase activity is blocked with 3% hydrogen peroxide in PBS (pH 7.45) with 1% Triton X-100 (TX) for thirty minutes, following which sections are washed in PBS three times for five minutes each and then treated with 88% formic acid for three minutes. After three rinses in PBS sections are taken into a blocking solution consisting of 1% goat serum in PBS for one hour. The primary antibody is then applied, 11A5, a monoclonal antibody against -synuclein phosphorylated at serine residue 129, overnight at four degrees C. After three washes in PBS, sections are incubated with biotinylated horse anti-mouse IgG (Vector Laboratories), diluted 1:200 for one hour at room temperature. Following three washes in PBS, sections are then treated with ABC (Vector Laboratories) for one hour at room temperature, washed in three changes of PBS and developed with DAB and hydrogen peroxide (Dako Cytomation) for three to five minutes. Following three rinses in 0.1 M Tris buffer, pH 8, sections are dehydrated through graded alcohols and xylenes.
Method 9: Sections are deparaffinized in Neo-Clear (EMD Chemicals), brought to distilled water through graded alcohols and immersed in 80% formic acid for 20 minutes. After washing three times in distilled water, endogenous peroxidase activity is suppressed by immersion for 30 minutes in 1% hydrogen peroxide in 0.1 M PBS with 0.3% Triton X-100, pH 7.4 (PBS-Tx). Sections are then washed three times in PBS-Tx as for all subsequent wash steps except where noted. Sections are incubated at room temperature overnight in LB509 antibody (Zymed Laboratories Inc., clone LB509, monoclonal mouse anti-human alpha-synuclein, Catalog #18-0215)at 1:1000 dilution in PBS-Tx. After washing in PBS-Tx, sections are incubated for two hours at room temperature in biotinylated anti-mouse IgG diluted 1:1000. After washing in PBS-Tx, slides are treated for 30 minutes with ABC (Vector Laboratories), with A and B components of the kit both at 1:1000 dilution. After two washes in PBS-Tx and a last wash in 0.05 M Tris buffer pH 7.6, slides are treated with DAB (5 mg/50 ml) and hydrogen peroxide (10l of 1% hydrogen peroxide/50 ml final solution volume) for 10-20 minutes in the dark (appropriate staining intensity is determined by monitoring a standard positive control section). Sections are then washed three times in Tris buffer, twice in distilled water and counterstained with hematoxylin. Lewy bodies and fibers are brown while nuclei and cytoplasm are blue. Slides are then taken through water, 70% alcohol, 95% and into 3 changes of 100%, 1 minute in each step. Tthe slides are then cleared with Neo-Clear and coverslipped.
Method 10: The method is depicted diagrammatically in Figure 6. Sections are deparaffinized in Neo-Clear (EMD Chemicals), brought to distilled water through graded alcohols and treated with 1:100 proteinase K (Enzo) at 37° C for 35 minutes (titration by concentration and time may be necessary). After washing three times in distilled water, endogenous peroxidase activity is suppressed by immersion for 30 minutes in 1% hydrogen peroxide in 0.1 M PBS with 0.3% Triton X-100, pH 7.4 (PBS-Tx). Sections are then washed three times in PBS-Tx as for all subsequent wash steps except where noted. Sections are incubated at room temperature overnight in LB509 antibody (Zymed Laboratories Inc., clone LB509, monoclonal mouse anti-human alpha-synuclein, Catalog #18-0215) at 1:1000 dilution in PBS-Tx. After washing in PBS-Tx, sections are incubated for two hours at room temperature in biotinylated anti-mouse IgG diluted 1:1000. After washing in PBS-Tx, slides are treated for 30 minutes with ABC (Vector Laboratories), with A and B components of the kit both at 1:1000 dilution. After two washes in PBS-Tx and a last wash in 0.05 M Tris buffer pH 7.6, slides are treated with DAB (5 mg/100 ml) with added saturated nickel ammonium sulfate (2 ml/100 ml) and hydrogen peroxide (5l/100 ml of 1% hydrogen peroxide) for 35-45 minutes in the dark (appropriate staining intensity is determined by monitoring a standard positive control section). Sections are then washed three times in Tris buffer, twice in distilled water and counterstained with 1% Neutral Red for 5 minutes. Lewy bodies and fibers are black while nuclei and cytoplasm are red. As Neutral Red is removed easily by alcohols, slides must be taken very quickly through water, 70% alcohol, 95% and into 100% with only 3 dips in each of the first 3 of these. After 3 minutes in two more changes of absolute alcohol, the slides are cleared with Neo-Clear and coverslipped.