I would first do a simple histological stain (H & E) to determine if tissue morphology is in order and then ascertain if you have a fixation or freezing problem. If the problem is freezing then as do a buffered sucrose cryopreservation step as suggested by Ditte, and as suggested by Gary check the state of the cryotome blade.

Why don´t you try performing thr cryosections WITHOUT previous fixation? Just freeze the sample in liquid nitrogen and the cut it directly in the cryostat. The immunohistochremistry should turn out even better, at least in my experience

Hi - also after PFA fixation you can store the tissue at 4oC in PBS (the tissue is fixed so should not decay any further).

I suspect the -80 step may be causing some problems.

Also "holes" or "artefacts" can be generated during the sectioning step if your blade is not sharp/clean. Use a new blade and move along it as you section to see if this helps

Thank you Jose i will try this also...

And thanks Gary..I have done sectioning earlier with the similar fixation (with old organs stored at liquid nitrogen) and i never had any problem but i think this is the freezing which is causing this problem..

 I would first do a simple histological stain (H & E) to determine if tissue morphology is in order and then ascertain if you have a fixation or freezing problem. If the problem is freezing then as do a buffered sucrose cryopreservation step as suggested by Ditte, and as suggested by Gary check the state of the cryotome blade.

Would you try to use scrioprotectors for your tissue? It could be 15-30 % sucrose or others, I think it will prevent tissue from destroing.

Thank You very much :) 

I agree wtih  all those who recommand cryprotection in 30% buffered sucrose. It is absolutly necessary if you are using PFA  fixied tissue.  The holes you are discribing are exactly that what Matthew calls the Peltier effect. H&E staining (Sheldon's recoammmandation) is very helpfull to get an impression of the quality of your tissue.

8% PFA seems to be a very strong fixation for immunohistochemistry. I recommand 4% PFA. in PBS pH 7.4  and if it is possible perfuse your animals.

Snap-freezing in liquid nitrogene is also a good idea but you must check your antibody weahter it works on native cryosections. Good luck!

I would like suggestions:

Thank you very much for your kind suggestioinb. They are really very helpful to me..

After testing what Gordon suggested, I would make some changes in the immunofluorescence protocol. Since you used 8% of PFA may have "masked" the epitopes, one way to overcome this problem is doing antigen retrieval. Another important point is to block the tissue autofluorescence.