Thick specimen can hardly be examined under light Microscope then,what is the purpose of staining it. Thick specimen or opaque specimens are better examined under scanning electron microscope SEM.

Hello Kartarzyna, it depends on the kind of your tissue. If you are work with compact tissue like brain, liver, kidney, spleen you can treat 20 to 50µm wtih a immunhistochmical protocol. Even here it depends on the protein you like to detect. The free floating method is very commen in neuroscience.

Here a short protocol:

use microtiter plates with 24 wells; each row of the plate is one working step.

1. Rinsing 3 x 10 min in PBS/TBS

2.Blockin of endogenous peroxidase (ony with Peroxidase containing detection systems).

3. Rinsing 3 x 10 min in PBS/TBS

4. Blocking of unspecific antibody binding (with normal serum or BSA) -- 90 min

5. Primary antibody (dilution has to be testet at first) -- overnight

6. Rinsing 3 x10 min PBS/TBS

7. secondary antibody biotiniylated or with Peroxidase or with fluorophores labeled -- 90 min

8.Rinsing 3 x10 min PBS/TBS

9.. StreptAvidin peroxidase or Fluorophore labeled -- 90 min

10. Rinsing 3 x10 min PBS/TBS

12. DAB-Staining for brightfield analysis 5-10 min

13. Rinsing 3 x10 min PBS/TBS

14. mounting on gelatine coated slides

13 dehydrating, clearing and mounting in a xylene soluble mounting medium (DAB)

or mounting with a water solubel mounting medium with or without Dapi for immunfluorescence

your dilution medium should contain 02.% Triton-X 100 and 1 % normal serum

The reaction should be processed on a rocking-table or shaker at room

temperatur. You can put your plate in the fridge but depending on the antibodies you will need higher concentrations.

Every antibody concentration has to be tested before use.

Good luck!