Immunohistochemical Staining of Biomarkers in Brain Tumor Neurosphere Sections

Abstract

Source: Núñez, F. J., et al. Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures. J. Vis. Exp. (2019).

This video demonstrates an immunohistochemical technique for labeling tumor biomarkers in paraffin-embedded brain tumor neurospheres. The neurosphere sections undergo paraffin removal and rehydration, followed by treatment to expose biomarker sites. Primary and secondary antibodies are then applied to label these biomarkers. The secondary antibodies are bound by avidin-conjugated peroxidase enzymes, which oxidize chromogenic substrates to indicate biomarker locations. Finally, nuclear counterstaining and slide preparation are completed for microscopic analysis.

Protocol

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.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rabbit polyclonal anti-ATRX,	Santa Cruz Biotechnology	sc-15408	IHC, 1:250 dilution
Rabbit polyclonal anti-Ki-67	Abcam	Ab15580	IHC, 1:1000 dilution
Rabbit polyclonal anti-OLIG2	Millipore	AB9610	IHC, 1:500 dilution
Goat polyclonal anti-rabbit biotin-conjugated	Dako	E0432	IHC, 1:1000 dilution
Vectastain Elite ABC HRP kit	Vector Laboratories Inc	PK-6100
BETAZOID DAB CHROMOGEN KIT	Biocare medical	BDB2004 L/price till 12/18