Beta Gal staining set from Roche is wonderful for frozen sections, however,
I do let the sections stain longer than 4 hours at 37C, overnight was
better and used inhouse prepared nuclear fast red as a counterstain.

Mouse lungs are perfused or is the correct term, infused? (filled with OCT)
via the trachea using a #18 hypodermic needle and no more than 1.5 mls OCT
to prevent blowing out the alveoli.  Cuts nicely at -20C, 5 um, but thicker
sections are desirable to insure seeing the Beta Gal reporter being
expressed. The mouse is not euthanized by cervical dislocation to prevent
destruction of trachea. Will be glad to describe this technic via private
email, more to it than described. 

Liver is always cut at -16C, warmer temp in cryostat prevents crunchy
sections with excellent  morphology.      

Fixation was a combination of formalin AND gluteraldehyde. 

My procedure was compliments of Robin Taylor, Genentech who clued me about
the Roche kit.  

There is a website which gives a BETA GAL "Bible" of sorts, excellent for
whole mount staining.  

We have opted to do whole mount beta gal staining the cryoprotect stained
lung, Cryojane tape transfer sections at 20 - 25 um.  This requires inhouse
prepared staining solutions. This was mainly due to not seeing the
expression in thinner, cryocut frozen sections using the Roche kit.
Location of staining in lung was like finding a needle in a haystack - our
particular problem.   


Gayle Callis
MT,HT,HTL(ASCP)
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)