Re: Mouse testes processing


I agree Teri, we started out using bouins for IHC with really good results, way
back in the day.

"Morken, Tim" wrote:

> Teri wrote:
> < concern is its effect on antigenicity.>>
> 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
> approx.
> 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!
> Teri
> 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
> legend!
> -----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
> profile.
>         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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