RE: [Histonet] mordanting in Bouin's, storage in 70%, processing mouse tissues

From:"Monfils, Paul"

Hello Elizabeth,

I'll do my best, based on my own experience.  Others may have different
insights ...

1. I don't know if Bouin's works "better", as I have never tried the
mercuric chloride mordanting.  But Bouin's works very well.  Bouin's is not
non-toxic by any means, but yes, it is substantially less toxic than the
mercuric solution. Sections can be immersed overnight at room temperature,
or an hour at 60 degrees.  I use saturated lithium carbonate in 70% ethanol
to remove the yellow color.  10 minutes is usually sufficient.

2. I'm sure there will be some difference of opinion here, but in my
experience tissues for morphological study can be stored almost indefinitely
in 70% alcohol.  Refrigeration is probably "best", but I have embedded and
sectioned tissues that have been stored in 70% alcohol at room temperature
for years, and gotten good results.  As I said, this is in reference to
tissues for morphological study.  Some specific substances may deteriorate
over time, though I haven't noticed any problems with most histochemical
stains on tissue stored in this way up to a year.  All of this of course is
assuming that the tissue was thoroughly fixed before it went into 70%

3.  This is a common problem with mouse tissue in particular, and
over-processing is the usual reason. Inadequate infiltration will certainly
make tissue difficult to section, but it won't make the tissue brittle.
Three changes of 100% alcohol doesn't sound extreme, but if the tissue
pieces are small (not a whole liver for example), you could probably get by
cutting the times in half.  Some people have experimented with adding a
small amount of glycerin to the absolute alcohol, and I'm sure folks will
have some other tips for you.

Paul M.

> ----------
> From: on behalf of
> Wurdak, Elizabeth
> Sent: 	Wednesday, June 21, 2006 12:21 PM
> To: 	Histonet (E-mail)
> Subject: 	[Histonet] mordanting in Bouin's, storage in 70%, processing
> mouse tissues
> Hello,
> I learned of this listserve group at the recent meeting of the Biological
> Stain Commission.  I wanted to join immediately because I teach histology
> to undergraduate students. We work through the whole paraffin technique in
> lab using manual methods.  We are always coming up with some questions.
> Here are a few:
> 1.  An  attendee at the Stain Commission meeting mentioned using Bouin's
> fixative  as a mordant prior to Mallory staining of formalin fixed
> tissues.  We have been using saturated mercuric chloride in 5% acetic
> acid.  Is Bouin's better?  Is it less toxic?  For how long should the
> sections be immersed in it?  Do you post-treat with lithium carbonate to
> remove the yellow picric acid before proceeding to the next step?
> 2.  How long can you store formalin fixed tissues in 70% alcohol?  Is it
> best to store them at room temperature or the refrigerator?
> 3.  Our mouse tissue blocks are coming out brittle, dry and hard to
> section with the exception of lung and testis samples.  I attributed this
> to incomplete paraffin infiltration, but lengthening the parafffin baths
> has not helped.  It was suggested to me that perhaps we are dehydrating
> too long.  We have been using 3 changes of a 100% alcohol for 1 hour each.
> What would you recommend?
> Many thanks in advance for your suggestions.
> Elizabeth
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