MAP-2 DAB staining problem

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zillem
zillem's picture
MAP-2 DAB staining problem

I want to stain Map-2 upon stroke using DAB staining for PFA fixed sections on slides, but instead of not staining the infarct, it is stained. I use a normal protocol for DAB staining:
1. Wash 3x 5min in 0,1 M PBS
2. 30 min blocking of endogenous peroxidase with 0,3 % H2O2 in 0,1 M PBS
3. Wash 3x 5min in 0,1 M PBS
4. 1h blocking in 5% serum (species 2nd AB), 0,3 % Triton X-100 in 0,1 M PBS
5. Incubation of 1° AB (MAP-2) overnight @4°C
6. Wash 3x 5min in 0,1 M PBS
7. Incubation of 2° AB (biotinylated) 1h @RT
8. Wash 3x 5min in 0,1 M PBS
9. Incubation of ABC Complex in 0,3% Triton X-100 in 0,1 M PBS 1h @RT
10. Wash 2x 5min in 0,1 M PBS
11. Wash 1x 5min in 0,05M Tris/HCl (pH 7.6)
12. Visualize with DAB for 2 - 10 min (10 ml Tris/HCl + 5 mg DAB + 6 l H2O2)
13. Wash 3x 5min in 0,1 M PBS
14. Wash 3x 5min in dH2O
15. Dry
16. Clear in Xylene
17. Coverslip with Entellan

I already checked the Map-2 in fluorescent microscopy, it works.

Has anybody an idea why it is the case?

Kristina Holmberg
Kristina Holmberg's picture
Hello,

Hello,

There are a few things that you can try to get it to work.
1. 30 min with H2O2 might be too much try a few shorter times eg 20 and 10 min. Because too much blocking of H2O2 starts chewing up the tissue and increases unspecific staining.

2. Preincubate with 10% normal serum specific for your secondary antibody, triton-X is not needed here.

3. Add 5% of the normal serum to all your antibody solutions.

4. Use 0.3% TritonX100 with your primary and secondary antibodies. But NOT with the ABC complex. The complex will not form well if you use triton-X.

5. Make the ABC complex first in 1 ml volume and after 30 min incubation add up to the final volume and incubate on your sections.

Good luck

zillem
zillem's picture
hi,

hi,
ok my mistake, we also checked 1., 3. and 4.

1. Wash 3x 5min in 0,1 M PBS
2. 30 min blocking of endogenous peroxidase with 0,3 % H2O2 in 0,1 M PBS (we checked also 10 min blocking)
3. Wash 3x 5min in 0,1 M PBS
4. 1h blocking in 5% serum (species 2nd AB), 0,3 % Triton X-100 in 0,1 M PBS
5. Incubation of 1° AB (MAP-2) in normal serum (+ Triton) overnight @4°C
6. Wash 3x 5min in 0,1 M PBS
7. Incubation of 2° AB (biotinylated) in normal serum (+ Triton) 1h @RT
8. Wash 3x 5min in 0,1 M PBS
9. Incubation of ABC Complex in 0,1 M PBS 1h @RT (we checked it also with 0,3% Triton X-100)
10. Wash 2x 5min in 0,1 M PBS
11. Wash 1x 5min in 0,05M Tris/HCl (pH 7.6)
12. Visualize with DAB for 2 - 10 min (10 ml Tris/HCl + 5 mg DAB + 6 l H2O2)
13. Wash 3x 5min in 0,1 M PBS
14. Wash 3x 5min in dH2O
15. Dry
16. Clear in Xylene
17. Coverslip with Entellan

so i will try 2. and 5., thanks!