[Histonet] low signal for long post fixation

[koellingr at comcast.net]

Mariela, 

(1) If you are looking at enzyme activity of a LacZ transgene to localize with X-gal staining, I found the situation really a problem with extended fixation times of vibratome sections (like you with controls or test article). Have never been able to "retrieve" that kind of staining. Maybe someone out there knows the trick. Especially bad in low-level integration of transgene. 
(2) I have though gone after the enzyme with just traditional IHC to B-galactosidase (there are good antibodies available. And as Rene said, increase your antigen retrieval which will (might, should) bring back the antigenic sites for IHC. But for the enzyme activity, I've never had much luck at that. 

Ray Koelling 
Faculty lecturer, WWAMI, UW School of Medicine, Spokane, WA 

----- Original Message -----

From: "Mariela Chertoff via Histonet" <histonet at lists.utsouthwestern.edu> 
To: histonet at lists.utsouthwestern.edu 
Sent: Thursday, March 23, 2017 6:28:24 AM 
Subject: [Histonet] low signal for long post fixation 

Dear all, 

I have mouse brain tissue perfused with PFA 4% and post fixed in formol 10% 
for periods between 2 month and 1 year. I cut them on vibratome (30um) and 
I made inmunohistochemistry and beta galactosidase to check senescence. 
My problem is that I have less signal on positive controls if postfixation 
on formol was longer. 

There is a way to solve this problem? 

Thanks in advance for your reply 

Mariela Chertoff, PhD 
Laboratorio de Neuroepigenetica - QB75 
Departamento de Química Biológica 
Facultad de Ciencias Exactas y Naturales - UBA 
Ciudad Universitaria Pabellón II Piso 4 
Ciudad Autónoma de Buenos Aires 
C1428EGA - Argentina 
Tel: 54 11 4576-3300/09 - Int. 221 
email:marielachertoff at gmail.com 
marielachertoff at qb.fcen.uba.ar 
<mariela.chertoff at uab.cat> 
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