RE: Mouse testes processing

From:"Monson, Frederick C."

OK, Teri,  then next you want to reduce the time in pHCHO and fix in the
fridge at 4oC.  I should have mentioned that all of my fixing is done at
4oC.  There are some exceptions, but not many and none for antigen

As an example, when I was doing urinary bladders, I did an experiment to
determine the effect of fixation on pressure exerted by the bladder.  What I
was looking for was the minimum time in cold pHCHO/PBS to kill and minimally
preserve the tissue.  The result of all of that was that I was satisfied on
many occasions to take tissue fixed for 10min in situ (vitro) and another
20min after removal.  Indeed, after the first 10 min fix, I could sample the
tissue without worry about shrinkage by contraction of the SM.  Now the
bladder is often thin, so that time may be inappropriate for another tissue,
but the principle is the same.  Fixing antigens for 20 hr is a long fix even
when done at low temp.  Further, auto processing works well for anatomy, but
one has to be very careful about time and temperature if one is trying to
preserve specific protein identifiers.  If I used 2 paraffin baths for an
hour each at 63oC rather than 60oC, I suffered irretrievable losses.   You
apparently have four of them to control.  And I have no idea what the other
substances are, but you must know that a large portion of the insterstitial
tissue is Leydig cell(s) and they are lipid-rich and most fixatives don't
preserve them well.  

Overcooking will shrink everything in a testis, and may leave what's left
looking OK but with a lot of space.  Further, this arftifact will vary with
the fixative used and the time of fixation.  

Any process that hopes to preserve antigenicity should be brief and
minimized.  I have taken tissues like the testis on a fast schedule and had
very good results.  Problem is that working with large numbers can make such
care very tedious.  If there is one antigen, then you should test for it.
There is no magic, just hard work.  Every Ag/Ab combo is an experiment
waiting to be done.


Fred Monson  

> ----------
> From: 	Johnson, Teri
> Sent: 	Wednesday, July 31, 2002 11:16 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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