Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

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1. Immunohistochemistry (IHC) of Paraffin-embedded neurospheres (NS)

1. Melt paraffin by placing the slides in a metal rack and incubating them at 60 °C for 20 min.
2. Deparaffinize the slides by incubation in a rehydration train at room temperature (RT): 3x xylene for 5 min, 100% 2x ethanol for 2 min, 95% 2x ethanol for 2 min, 70% 1x ethanol for 2 min, and 1x deionized water for 2 min. Hereon, keep the slides in tap water until antigen retrieval, as drying out will cause non-specific antibody binding.
3. Incubate the slides in citrate buffer (10 mM citric acid, 0.05% non-ionic detergent, pH 6) at 125 °C for 30 s and at 90 °C for 10 s. Let them cool down, then rinse the slides 5 times with distilled water.
4. Incubate slides at RT in 0.3% H₂O₂ for 30 min.
5. Wash the slides 3 times in washing buffer (0.025% detergent in Tris-buffered saline [TBS]) for 5 min with gentle agitation.
6. Incubate the slides at RT with blocking solution (10% horse serum, 0.1% bovine serum albumin [BSA] in TBS) for 30 min.
7. Incubate the slides overnight at 4 °C in a humidified chamber with primary antibody prepared in dilution solution (0.1% BSA in TBS). Wash the slides 3 times in washing buffer for 5 min with gentle agitation.
8. Incubate the slides at RT for 1 h with a biotinylated secondary antibody prepared in dilution solution. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.
9. Incubate the slides at RT in the dark for 30 min with avidin-biotinylated horseradish peroxidase complex. Wash the slides 3 times in washing buffer for 5 min with gentle agitation.
10. Incubate the slides with 3,3′-Diaminobenzidine (DAB)-H₂O₂ substrate brands here for 2-5 min at RT.
11. Rinse 3 times with tap water. Dip (1-2 s) the slides in hematoxylin solution to counterstain them, then rinse them thoroughly in tap water until clear.
12. Dehydrate the slides as follows: incubate in distilled water for 2 min, dip 3 times in 80% ethanol, incubate in 95% ethanol 2 times for 2 min each, incubate in 100% ethanol 2 times for 2 min each, and finally in xylene 3 times for 3 min each.
13. Coverslip the slides with a xylene-based mounting medium and let them dry on a flat surface at RT until they are ready for imaging. 

NOTE: If performing DAB staining, the slides can be stored at RT. However, if immunofluorescence staining is performed, slides should be stored in the dark at -20 °C to reduce photo-bleaching.

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