[Histonet] RE: Frozen section fixing problems - but paraffin works!

[C.M. van der Loos]

Dear Tan,
Your protocol for staining the cryostat sections needs some adaptions 
indeed.
First, cut your sections and allow them to dry at least 1 hour at RT. 
Preferably longer, for example over night under a fan. Next, fix your 
cryo's in pure acetone for 10 min at 4C, let them air-dry. Block 
endogenous peroxidase activity with 0.3% perhydrol + 0.1% Na-azide in 
TBS or PBS (20 min, RT) and wash with TBS or PBS (3 x 2 min). Then, 
start with the donkey serum etc. and your usual staining procedure.

Some comments:
You should avoid the use of alcohols or methanol in the staining 
procedure, because most antigens will be damaged by this. 
Blocking with 0.3% peroxide (in buffer I hope??) is not recommended 
because the formation of air bubbles that may damage your tissue 
section. The addition of Na-azide prevents the formation of air bubbles 
and also acts as inhibitor of peroxidase activity.

Good luck with staining!
Chris van der Loos
Dept. of Cardiovascular Pathology
Academical Medical Center
Amsterdam - The Netherlands


----- Original Message ----- 
>From  "Tan, MinHan" <MinHan.Tan <@t> vai.org> 
Date  Wed, 19 Nov 2003 17:32:59 -0500 
To  <histonet <@t> lists.utsouthwestern.edu> 
Subject  [Histonet] Frozen section fixing problems - but paraffin 
works! 

Hi,
I am working with a new monoclonal antibody in parathyroid tissue, and 
I use the ABC-DAB reageant system for immunohistochemistry.
 
I am new to frozen sections immunostaining - and I am encountering 
problems with frozen sections - staining is very weak or absent, in 
comparison to paraffin embedded tissue, which I have no problems with. 
 
My protocol for frozen sections: 
Unfixed frozen slides with 5 micron sections thawed at room temperature 
for 5 minutes
Placed in 70% ethanol x 5 minutes.
Washed in PBS x 5 minutes.

followed with standard protocol: hydrogen peroxide 0.3% x 30 min, 
donkey serum 5% x 30 min, primary antibody (mouse) 4 deg overnight, 
secondary (goat anti-mouse), ABC, DAB.
 
Does anyone have any advice?
Thank you!

Regards,
Min-Han Tan