Dear Histonetters,

I had a researcher come into my lab with a protocol for Beta-Galactosidase on
rabbit hearts.  He wanted my help in trying to improve upon the protocol so
that
he could produce photographic quality sections, which he felt he was not
getting.  According to this protocol, he is supposed to wash the fresh sections
briefly in concentrated sucrose to increase the osmolarity of the tissue, thus
removing the water from it and causing less cracks.  Then, he slowly
freezes the
tissue directly in liquid nitrogen, and cuts 10 - 15 micron frozen
sections.  He
fixes the sections in 10% N.B.F. for 2 minutes, washes them in PBS, and
incubates them in his Beta-Gal solution in the 37 degree oven until he sees
blue
form on the sections macroscopically ( I am not sure what the time range is ).
He quickly stops the reaction with PBS to prevent an over-reaction,
counterstains for 20 seconds with Eosin, dehydrates in graded alcohols, clears
in xylene, and coverslips.

 Since I have never done this procedure before, I figured the best thing to do
was to try to increase the morphology of his tissues and improve the quality of
sectioning.  Since this sucrose wash and slow, direct freezing in liquid
nitrogen have been no-no's in my histoworld, I told him to quickly freeze the
tissue in isopentane cooled in liquid nitrogen.  He brought me the frozen
tissue, which sectioned beautifully at 5 microns.  However, when we ran the
rest
of the protocol, we could not get a reaction.  He kept wondering if the
isopentane somehow interfered with the Beta-Gal reaction.  I, however, have
never had any problems with isopentane before, and realized that there are too
many variable to name.  Could it be the thinner micron sections, the wrong
pH of
the PBS ( I use pH 7.2 ), for example.

Can someone out there give us their protocol and give us some insight into
variables associated with this enzyme?  Is there a protocol out there for
Beta-Gal on formalin-fixed, paraffin processed sections?   Thanks in
advance for
help!

Sincerely,
Suzy Winden, HT
Experimental Surgery Pathology Core Lab
Duke University Medical Center
Durham, NC