Immunohistochemistry Procedures
Paraffin Sections
1.Prior to performing the IPOX (immunoperoxidase) experiment, dewax the paraffin sections by baking them at 60 degrees C for 30 minutes and then putting them through citroclear [Citroclear is a mounting agent (chemical name Limonene, also known as Histoclear, Bioclear)].
2.Hydrate the sections through the following series:
A.3 X 5 minutes xylenes
B.3 X 5 minutes 100% Etoh
C.2 minutes 95% Etoh
D.2 minutes 70% Etoh
E.1 minute 50% Etoh
F.1 minute ddH2O
G.1 minute TBS
1.Block endogenous peroxidase with 0.5% hydrogen peroxide in water, for 30 minutes.
2. Antigen unmasking is performed by incubating at 60 degrees C for 16 hours, in 50mmol/L Tris and 0.2 mmol/L EDTA (pH 9.0), using a covered water bath.
1.Rinse slides with PBS and then incubate with PBS containing 0.2% Triton X-100 for 10 minutes.
2.Rinse slides with PBS.
3. Incubate sections with 1:8000 dilution of anti-HIF-1 alpha (NB100-131) for 90 minutes at room temperature (RT).
4. Incubate sections in secondary HRP-conjugated goat anti-mouse serum for 30 minutes at RT.
5. Incubate sections in tertiary HRP-conjugated rabbit anti-goat serum for 30 minutes at RT.
6. Develop the peroxidase reaction using diaminobenzidine.
7. Wash slide and mount in aqueous mountant.
Substitution of the primary antibody with PBS can be used as a negative control.
1. Deparaffinize to water:Xylene #1-10 dipsXylene #2-10 dips 100%EtOH #1-10 dips 100%EtOH #2-10 dips 95% EtOH-10 dips 70%EtOH-10 dips diH2O-2 changes
2. Rinse in PBS for two minutes.
3. Quench slides is MeOH/H2O2 for 5-10 minutes (1 part 30% H2O2/36 parts 70% MeOH; 8 mls H2O2/288 mls 70% MeOH).
4. Unmask antigens by boiling for 3 minutes in 0.01 M Citrate Buffer, pH 5.5. 47.2gr Sodium Citrate 8.3gr Citric Acid pH to 5.5 qs to 0.5 L dH2O
5. Rinse in PBS.
6. Apply 2 drops blocking solution (10% non-immune normal goat serum, Zymed Labs, Cat # 50-197). Incubate for 10 minutes in humidity chamber
7. Incubate for 10 minutes in humidity chamber.
8. Do not rinse.
9. Incubate in mAb HIF-1 alpha (cat# NB 100-131), diluted 1:250 in PBS (10ul /2.5mls) overnight at 4 degrees C, in humidity chamber.
10. Rinse in PBS.
11. Incubate in 2 drops Biotinylated Secondary Antibody for 10 minutes in humidity chamber.
12. Rinse in PBS.
13. Incubate in 2 drops Enzyme Conjugate solution (HRP-Streptavidin) for 10 minutes in humidity chamber.
14. Rinse in PBS.
15. Incubate in 2 drops Substrate-Chromatogen solution AEC solution, (AEC Single Solution, Zymed Labs, Cat# 00-1111) for 5-10 minutes in humidity chamber.
16. Rinse well in dH20.
17. Counterstain with hematoxylin for 1 minute.
18. Rinse well in tap water until it runs clear.
19. Mount coverslip with water soluble mounting media. Do not dehydrate. (Alcohols will remove the AEC color).
