--On Wed, Nov 4, 1998 12:56 pm -0500 "Katherine Terry" <[log in to unmask]>
wrote:

> I'm a grad student starting project involoving confocal study of
> neuropeptides in the brain of the insect Rhodnius prolixus.  I'm trying
to
> decide what type of fixative to use on the whole mounts I plan to
prepare.
> Most literature suggests using formaldehyde (2-3%) in a buffer.  Some
> epifluorescence papers I've read use 'a modified Bouin-Hollande
solution'
> including 0.7% mercuric chloride and lacking acetic acid.  I've never
seen
> this solution used for confocal studies.  Is there a reason for this?
> Also, I've talked to a few people who have suggested using 0.1M glycine
to
> quench autofluorescence of formaldehyde after fixing.  Does anyone have
a
> reference which might explain the virtues of this?  Is it even
necessary?
> One last thing (I promise!) -- I've also been digging through papers
> looking for a mounting media which will reduce photobleaching.  Things
> I've come across include DABCO, and something called Fluoroguard from
> Biorad.  Can anyone offer information about the performance of these
> antifades?
>
>                                         Thanks!
>
>                                                 Kathy T.


Hi Kathy,

for whole mounts, people usually use 4% paraformaldehyde in 0.1 M PB.
This should be perfectly fine for neuropeptides in insect brain
wholemounts, and also for frozen sections.  Personally, I would use
Bouin's, Bouin-Hollande (with or without acetic acid), Boer's (picric acid
sat. aqu. sol : 25% glutaraldehyde = 3 : 1, + 1% acetic acid), or any
other Bouin variant only for paraffin sections (which might work very well
for neuropeptides).  Paraform/PB is usually bad for paraffin sections.

I once experimented with Bouin's and fluorescence (Lucifer Yellow/FITC and
TRITC filters).  The picric acid quenched all fluorescence, i.e. including
tissue autofluorescence.  Once the tissue was paraffin sectioned, and the
picric a. completely removed from the sections, fluorescence was back.
However, getting all the picric acid out of a wholemount will take
extended washes, whereas in sections it comes off automatically during
rehydration (i.e. I do not bother washing all the picric a. out before
embedding in paraffin).

I don't think there are many papers (I don't know a single one) where they
recommend Bouin's for whole mounts.  Bouin's somehow always goes with
paraffin.  However, if your antigen likes picric acid, you could use
Zamboni or Stefani(ni?), which is basically paraform/PB + picric acid, eg:
0.1 M PB, 4% paraform, 7.5% picric acid sat aqu. soln., or
0.1 M PB, 2%    "    , 15%            "

For fluorescence, you would still have to thoroughly get rid of the picric
acid afterwards.  Also, my observation was only in sections (10um);  it
could be that, for thick specimens, the picric acid that is actually bound
to the tissue and that you thus cannot wash out, quenches fluorescence at
least partially.  Maybe somebody else on the list might know more on this.

A useful reference is
Naessel D.R. (1996) Advances in the immunocytochemical localization of
neuroactive substances in the insect nervous system.  J. Neurosci. Methods
69:3--23.

Let me know how you're getting on,
                                    Swidi

PS: I'm a locust brain stainer, by the way...

--------------------------------------------------
Swidbert R OTT
Department of Zoology
University of Cambridge
Downing Street
Cambridge CB2 3EJ
ENGLAND