Immunohistochemistry Protocol for MAP1B Antibody (NB600-1446)

IHC protocol for cryostat sections (Protocol generously provided by Dr. Yimin Wang, Tissue Regeneration Therapeutics Inc )

1. Take out the cryostat section slides from -80C. Dry for few minutes at RT.
2. Rinse slides with PBS to get rid of OCT. 3 min x 2, shake
3. Fix slides with 1:1 methanol:acetone for 3 min
4. Wash slides with PBS, 3 min x 2
5. Circle tissue sections with PAP-Pen,
6. Incubation with Normal Horse Serum: 5% in 2%BSA (in PBS-Tween20) for 20 minutes.
7. MAP1B anti human antibody (1:200 in 2% BSA 0.1% Tween20). Incubate for 1.5 hours at 37C in a humid chamber.
8. Wash slides with PBS-Tween (0.1%), 5min x2

The following steps are for IHC only

9. Blocking of endogenous peroxidase: Incubation with 0.3% H2O2 for 10 minutes.
10. Wash PBS-Tween (0.1%), 3 min x 2
11. Incubation with secondary antibody (1:200 with PBS, anti mouse-bio, in AK-5002 kit) for 45 minutes at 37C in a humid chamber.
12. Wash slides with PBS-Tween (0.1%) 5min x2
13. Incubate with VECTAINABC reagent solution (bottle C, Cat No: PK6100, contains Avidin-HRP) for 30 minutes at RT in the dark.

(For solution C: 10ml PBS + 2 drops of A and 2 drops of B, kept in 4C with dark)

Prewarm at RT for 10min before use and drop 2 drops to the sections.

14. Wash slides with PBS-Tween (0.1%) 5min x2
15. Incubation with peroxidase substrate (Vector Nova Red, SK-4800) until desired stain intensity develops. Usually 1 to 2 minutes.

Prepare the substrate immediately before use: can be used for > 1week.

- Add 3 drops of Reagent 1 to 5 ml of DDW, mix well

- Add 2 drops of Reagent 2 and mix well

- Add 2 drops of Reagent 3 and mix well

- Add 2 drops of Hydrogen Peroxide Solution and mix well

16. Wash slides with tap water for 5 minutes.
17. Hematoxylin nuclear staining: (Filter solution when first use, HE staining Jar,2nd one). Stain slides for 5 seconds at room temperature, then carefully rinse for 3 minutes under running cold tap water;
18. Mount the slide with cc mount (Sigma, C9368, 30ml)