RE: cutting whole mice

From:Robert Geske

Not exactly Patsy,

i agree that frozen sections need to be used for the enzyme histochemical
method for the demonstration of -galactosidase.  we do a considerable
amount of gene expression studies on mutant mouse lines that carry the LacZ
gene as a selection marker.  for this we do standard ABC IHC on formalin
fixed (Zinc formalin although I expect 10% NBF would be fine) on paraffin
sections.  the antibody we use is a rabbit polyclonal from Cortex Biochem.
The same antibody used to be available from 5' 3'.  We use it at 0.3g/ml
for 30 minutes on a Ventana Discovery --- 

I really do not understand the original message in this thread ---"He is
injecting them with Beta-Gal at birth, then wants to look for the
stain......"  is he injecting them with the enzyme ??  what "stain" is he
looking for and where is the substrate coming from??  is he actually
injecting the X-gal solution into animals that  are making -galactosidase
because of either genetic alteration of embryonic stem cells or adenovirus
insertion ?? And if he is injecting the X-gal solution into neonates, I
would be interested to know the mortality rate after these injections.  Not
to get bogged down by symantics but the original message states "whole
mounts" and then goes into requesting sections  --- which does the
investigator want? The fixed animals can easily be submerged into the x-gal
staining solution, we have done this, but i do not have experience with
doing frozen sections on whole mice --- there was actually a Leica sales rep
who had considerable experience with this and would be a great resource to
tap. The original post asked for a reference --- I would suggest "Short
Protocols In Molecular Biology"  -- its a good nuts and bolts reference to
have around.  


If we could get additional information from the originator, we might be able
to answer the question more directly.

rob

-----Original Message-----
From: rueggp [mailto:rueggp@earthlink.net]
Sent: Monday, November 26, 2001 5:27 PM
To: Jamie Erickson
Cc: histonet@pathology.swmed.edu
Subject: Re: cutting whole mice


thanks Jamie,
that is what i thought.  not sure if they want to use the beta-gal enzyme or
ihc method but either way i think the tissue needs to be frozen, in my
experience anyway.
patsy

Jamie Erickson wrote:

> Patsy,
>         We've only do B-galactosidase staining on frozens. I don't know if
this is what your asking but we treat mice with adenovirus that express the
marker  gene B-galactosidase then freeze the tissues  (after days of
injection of the virus ) for staining and using the enzymatic method (X-gal,
Ferric and Ferro cyanide etc..) and detect B-gal in various tissues. I have
not used the B-gal antibodies for B-gal staining, the enzymatic method is a
lot easier. Hope this helps..
>
> Jamie Erickson
> Scientist II
> Wyeth/Genetics Institute
> 1 Burtt Rd.
> Andover, MA   01810
> work : (978) 247-1348
> FAX  : (978) 247-1389
> Jerickson@genetics.com
>
> >>> rueggp  11/26 5:35 PM >>>
> fellow netters,
> i have been asked about cutting whole young mice treated with beta-gal.
> i did this at one time as frozen sections by placing a microtome in a
> chest freezer and using a tungsten carbide knife.  question:  in order
> to sucessfully do ihc for beta-gal would the sample have to be frozen or
> does it stand up to processing?
> the exact inquiry follows.
> patsy
> Dear Patsy,
> I was given your name by Nancy Shellhorn and she thought you would be
> just the person to help me!
> I work in a core lab which provides histology support to many users.  I
> have an investigator who is interested in doing whole mounts of young
> mice.  He is injecting them with Beta-Gal at birth, then wants to look
> for the stain after they have grown a little- he says they will be
> "young mice" but hasn't given me an exact age.  I believe weeks as
> opposed to months.
> Have you any experience processing entire small mice and cutting them?
> If not, can you advise me of a source (human or literature) that may be
> able to assist me?
>
> Thank You Very Much,
> Marda Jorgensen



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