RE: Mouse testes processing

From:"Morken, Tim"

Teri wrote:

Actually bouins's is a very good fixative for perserving antigen. 

Tim Morken
CDC, Atlanta

-----Original Message-----
From: Johnson, Teri []
Sent: Wednesday, July 31, 2002 11:17 AM
To: Monson, Frederick C.
Cc: List-HistoPath
Subject: RE: Mouse testes processing

Fred, et al,

Thanks for your help and suggestions.  In this case, the tunic was excised
before fixation took place.  The specimens were in 4% paraformaldehyde for
20 hours before being placed in 70% alcohol.

I don't have a problem using Bouin's fixation for these specimens, but my 
concern is its effect on antigenicity.  I'm thinking of trying zinc formalin
next, just to see what effect, if any, it has on the connective tissue.

I'm open to any other suggestions you may have!


P.S. It must be fun watching all the male rodents run to the farthest
corners of their cages when you arrive, your skill at gonad dissection is

-----Original Message-----
From: Monson, Frederick C. []
Sent: Wednesday, July 31, 2002 9:57 AM
To: Johnson, Teri
Cc: 'List-HistoPath'
Subject: RE: Mouse testes processing

Morning Teri,
	As a young graduate student, I fumed over the male mouse gonad (rat
too!), and its apparent wish to become something quite unintended following
most fixations and paraffin embedment.  It was clear that there was a REAL
barrier to diffusion of the fixative components that lay in the thinness of
the tunica albuginea.  So, I, desiring to cut through this impediment, began
to bisect the organ (but only like Mostly Headless Nick!) so related halves
could be re-approximated in the block.  This had widespread and salutary
effects both on the quality of my histology and also on  my psychological
	Later, when I decided to preserve the tubular mass intact but sans
tunica, I resorted to the following.  With a pair of iris scissors and #5
Dumont forceps, I would grab the tunic and then cut it along an equatorial
line at least as long as 1/3 a circumference.  Then I would grab an end of
the tunic with the forceps and with another pair would clamp them over a bit
of tunic only held by the first pair.  With the second pair closed, but not
on the tunic, I would gently draw the tunic through the space between the
tines of the second pair by separating both pair of forceps.  In the freshly
excised mouse testis, the tunic comes away without much resistance.  In the
rat the same occurs with somewhat more resistance, and in the gerbil and
hamster similar results.  Guinea pig also works easily.  I found this method
of stripping the tunica albuginea very helpful in exposing the tubules and
interstitial tissue en masse without much evidence of damage.
	At the very least in my experience (400+ animals), the tunica must
be cut or no matter the processing  the smooth surface of the tunica will be
dimpled or caved in some part of its surface.  The most bothersome aspect of
working with testis is the differential effects of fixing and subsequent
paraffin processing on the relative volumes of tubule and/or insterstitium.
I have always checked the temperatures of the paraffin baths before
processing testis, and I have always been very attentive to the times in
absolute ethanol and in xyleme or benzene.  This attention has helped to add
some uniformity to the processing schedules I used on my preps.
	The myoid layer on the surface of each tubule is myogenic and
sensitive to oxytocic {octa-peptides (How many amino acids? [One of the
great biochemistry questions!])}.  The contractions of isolated tubules in
plain saline can be sufficiently strong to expel the seminiferous epithelium
completely intact from 1" segments.  With oxytocic stimulation one simply
augments such behavior.  This myoid layer can be seen to undulate with
predictable frequency that is sensitive to temperature and hormonal
conditions.  In any case, these cells, if not fixed rapidly, could affect
tubular volume in some areas of the organ.
	Hope this helps,

Fred Monson

> ----------
> From: 	Johnson, Teri
> Sent: 	Tuesday, July 30, 2002 11:21 AM
> To: 	Histonet (E-mail)
> Subject: 	Mouse testes processing
> Hello all!
> I'm having difficulty with routine processing of mouse testes.  I haven't
> been able to preserve the intertubular connective tissue.  I've tried
> bouins fixative and it's well preserved.  It mostly disappears with 4%
> paraformaldehyde and 10% neutral buffered formalin.  The simple answer is
> to use bouins, but what I'd really like to do is figure out how to
> preserve it with routine fixation/processing.
> My processor is set up as follows:
> 70% alcohol
> Prosoft x5 stations
> ProPar x3 stations
> paraffin x4 stations
> I will post a picture to the site and will send another email
> with the path when it's posted.
> Also, for those of you using Davidson's fixative on mouse tissues, have
> there been many problems with subsequent immunostaining?
> Thanks for your help,
> Teri Johnson
> Managing Director Histology Core Facility
> Stowers Institute for Medical Research
> 1000 E. 50th St.
> Kansas City, Missouri  64110

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