Dear All,

   I have a problem with IHC staining(using Perioxdase,AEC substrate)) on 30 um  thick mouse lymph node  frozen sections. After staining part of section is  washed away and the morphology of the section is poor. Can  someone help me?

 My protocol is following:

                               IHC  staining protocol

1. Cut 30 um frozen sections.
2. Fix in cold methanol  for 3 minutes, then in cold aceton for 3 minutes.

Air dry for 1 hour.

3. Put the section in PBS for 10 minutes.
4. Block with 1%FCS/PBS  100 ul for 20 minutes at room  temperature.
5. Endogenous biotin blocking:

(1)   Incubate slice in egg solution for 15 minutes.

Egg solution: 25 ml egg white ,75 ml distilled water.

Wash with 2 changes of  distilled water.

(2)   Incubate slice in the milk(fat free) for 15 minutes at room temperature.

   Wash with 2 changes of distilled water.

6. Add diluted first antibody.

For example :CD3-biotin

Incubate 2 hours at room temperature or at 4 °C   overnight.

Wash in PBS for 30 seconds, 3  minutes and 10 minutes three times.

1. endogenous peroxidase blocking:

The solution : 0,3%  H2O2  ,0,25% NaN3   in PBS

 Incubate the section in this solution for half  hour.

 Rinse with water for 2 minutes.

 Wash in PBS  for   5 minutes.

8        Block with 1%FCS/PBS for another 20 minutes at room temperature.

9. Add   second antibody

   for example streptavidin-PO

      Incubate for 4 hours at 4 °C..

     Wash with PBS for 1 ,3 ,10 minutes for three times.

9        Add substrate 300 ul for each section, incubate at 37°C surface for half  hour.

Substrate solution:    25 ul AEC stock solution

                                  1 ml Na-Acetate buffer(5,4)

                                   1ul H2O2

Attention: Make this solution just before use,protect it from light..

Wash in PBS for 30 seconds,3 minutes   and 10 minutes for three times.

10    Dry on 37°C surface for 5 minutes.

11      Mount with  175 ul Kaser’s galatine using 24x60 cover  glass.

12    Read the slides.

 Will  adding Triton  or tween 20  help ?

 Thank you.

Bin ma

German National research Centre ofBiotechnology

Braunschweig

Germany