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Author Topic: MAP-2 DAB staining problem  (Read 4058 times)

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Offline zillem

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MAP-2 DAB staining problem
« on: March 24, 2008, 07:15:01 AM »
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, such as this one: http://www.histochem.net/histochemistry%20protocol%20avidin-biotin.htm
I already checked the Map-2 in fluorescent microscopy, it works.

Has anybody an idea why it is the case?

MAP-2 DAB staining problem
« on: March 24, 2008, 07:15:01 AM »

Offline gula

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Re: MAP-2 DAB staining problem
« Reply #1 on: March 24, 2008, 10:22:41 AM »
In this protocol I see no step of blocking endogenous biotin. Is it possible, that your tissue is rich in biotin? That would make a false positive reaction.
gula

Offline zillem

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Re: MAP-2 DAB staining problem
« Reply #2 on: March 24, 2008, 10:31:26 AM »
I only block the endogenous peroxidase and with the serum of the species of the secondary AB, the biotinylated one.

The exact protocol is like that:

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 @4C
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
« Last Edit: March 26, 2008, 07:19:08 AM by zillem »

Offline gula

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Re: MAP-2 DAB staining problem
« Reply #3 on: March 24, 2008, 02:03:15 PM »
Yes, there is no step of blocking endogenous biotin. You can find such protocol in the protocol sector of ihc-world.
Biotin is usually rich in kidney, liver, bowel and lymphatic tissue. I would recommend to try this blocking step and look again.
gula

Offline zillem

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Re: MAP-2 DAB staining problem
« Reply #4 on: March 24, 2008, 03:05:57 PM »
Hum, the only thing I want to argue is, that the protocol did work @sb else who gave it to me... And I want to stain brain sections, but I will try another blocking step as you recommended to me. Thx.

Offline CanuckPhD

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Re: MAP-2 DAB staining problem
« Reply #5 on: March 25, 2008, 09:33:06 AM »
An issue may be that you have many lysed RBC in your infarct area. This of course would result in lots of peroxidases (thus the reason you didn't see any issues with your fluorescent staining). An idea to check if this is the case is to do your peroxidase blocking step, wash and then place your slides in DAB solution. If positive then this is the issue. If so try using a PBS/Na azide/1% H2O2 for 30 minutes, as this may be more effective then your current method.

When I have performed DAB iHC previously on stroked rats this seemed to work.

Good luck.

Re: MAP-2 DAB staining problem
« Reply #5 on: March 25, 2008, 09:33:06 AM »