Robbin,
A research fellow and myself are doing experiments using different variables on
beta gal as well.  I have had some very helpful entries on the histonet, and
Gail Callis is sending me some extensive information on beta gal.  Please
let me
know how your experiment turns out and I will do the same.

Thanks,

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







"Robbin Newlin Manager, Histology Core Facility" <rnewlin@burnham-inst.org> on
02/01/2000 01:30:46 PM

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 To:      winde002

 cc:



 Subject: Re: Beta-Galactosidase







winde002@mc.duke.edu wrote:
>
> 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
Hi Suzy
I to have seen the same problem and I would be very interested in any
replies that you get on the subject. I had some mouse organs that I
devided into two parts, one half I did a wholemount stain on and the
other half I froze and cut 10 micron sections and did beta gal staining.
The wholemounts were screaming with beta gal (very suspicious) and the
sections had significantly less staining but it was in all the rights
places. The post doc of course went straight for the wholemounts and
claimed that the sections were not stained properly. (whatever) I would
like to get to the bottom of this problem, I am currently setting up an
experiment to test the freezing method. I have never been able to get
beta gal to work in paraffin sections, I have also not been able to find
a good antibody to use.
Thanks for listening
Robbin