[Histonet] TUNEL on FIXED rat PUPS

[Guillermo Palchik]

Dear Histologists...

I am currently doing an experiment in which I perform TUNEL on brain  
slices that were treated with certain drugs, to examine the levels of  
apoptotic cell death. The rat’s brain is flash frozen (scooped from  
the skull, directly into cold isopentane) air dried, and then stored  
into a -80 C freezer.  We then cut the brain into 20 um slices using a  
cryostat and mount them on slides, and store them in a -20 C freezer,  
where they remain until the TUNEL step. The TUNEL is performed using  
the Apoptag Plus kit from Chemicon using Peroxidase/DAB (Cat. # S7101)  
and counterstaining with 0.5% methyl green. This has been done in the  
lab for quite some time now and we are able to get results…
We want to switch over to perfused brains (instead of flash frozen).  
However, we have had ZERO positive staining once the brains are fixed  
(in 4% PF). This has been corroborated by other lab members that have  
tried it for a couple of years already…
The protocol that came with the kit has a section for flash frozen and  
a section for fixed tissue, and since I have done TUNEL using fixed  
tissue before, I know that it is possible to do TUNEL in fixed tissue,  
however we cannot get any positive staining whatsoever… Along these  
lines, since the first step of the Apoptag TUNEL protocol is to fix  
the tissue with 4% PF (for 10 min), this has led me to believe that  
the problem is indeed the initial fixation with PF (at the time of  
perfusion).
I should say that we work with rat PUPS for this and that the immature  
brain is not the same as the mature brain (the immature brain has more  
fat, for example) and that this might be the cause of the problem…In  
any case, I was doing some research and I wanted to try using Zinc  
Formalin as a perfusate and see if this would allow us to do the TUNEL.

I would appreciate any comments and suggestions regarding this. I am  
sorry for the lengthy email, but I wanted to show a more or less  
complete picture, in case I am overlooking other factors…

Guillermo Palchik
gp62 <@t> georgetown.edu