Hello Everyone

There has been a lot of interest on and off the list in my comments
yesterday about processing tissue for immunolabelling without the use of
detergents. We are mainly a neuroscience lab so the antigens we are
interested reflect that. We deal almost entirely with intact tissue (ie
not cultured or isolated cells). What follows is a reasonably detailed
description along with some history, (so that you don't get the idea I
worked this all out by myself!)


FIXATION
For light microscopy, our routine fixation is Zamboni's fixative
(originally designed for fixing sperm for TEM!). This is 0.2% picric
acid and 2% formaldehyde in 0.1M phosphate buffer at pH7.0. It works for
a very wide range of antigens including cytoplasmic enzymes, nuclear
markers, cytoskeletal elements, membrane bound proteins, neuropeptides,
cell surface receptors etc. It also fixes well other intracellular
labels such as biocytin / neurobiotin, DiI, Fast Blue etc. For TEM
immunolabelling, we add 0.05%-0.1% glutaraldehyde, (and sometime
increase the formaldehyde to 4%) which gives really excellent EM
preservation (see Murphy et al., J. Comp Neurol 398, 551-567, 1998).


PERMEABILISATION
Our approach to this began back in the late 70's with initial work by
John Furness and Marcello Costa here at Flinders, who worked out how to
do whole-mount immunofluorescence on the gut. They found that detergents
really produced a high backgorund, unreliable penetration and a lot of
spurious artefacts. So they developed a procedure based on tissue
"clearing" where the tissue is dehydrated though ethanols to xylene,
back through ethanols to PBS. The idea here is that the xylene made the
holes in the membranes to let the antibodies in - it works a treat and
we still use this protocol when we are making whole mounts of tissue
with a lot of dense or fatty connective tissue.

Sometime in the early 80's, a paper from Llinas' lab reported the use of
DMSO as a clearing and permeabilising agent that almost totally
eliminated tissue shrinkage. We confirmed that this was true and
developed a range of protocols based on using DMSO. The protocols seem
pretty robust and different labs around use slight variants that have
evolved with time.

The simplest method involves washing out the fixative of the tissue
(either chunks of tissue or vibratome sections or whatever) with PBS
followed by 50% (or 80%) ethanol, then washing a few times in DMSO then
back into PBS. Some labs here use a closer series of ethanols, some just
go straight from PBS to DMSO and back. We have looked at single dye
filled neurons (intracellular injection of either fluorescent dextran
or Alexa-biocytin) right through the fixation / DMSO process and we have
confirmed that there is no detectable change in cell dimensions at any
stage.

For EM processing, we wash the fixed tissue sections (25 - 50 microns
thick, prepared in various ways: Vibratome; gelatine embedding etc) in
50% ethanol, as originally developed by Ida Llewellyn-Smith years ago.
(gelatine embedding as a way of getting sections of fixed bits of small
tissue blocks is in the Murphy et al ref cited above).

With any of these protocols, we get antibody penetration of up to about
25-50 microns from the surfaces, which is fine for most purposes
(although we usually don't go over 30 micron thick sectionns, bu we do
get good penetration through whole mounts up to 100 micron thick...)

The other thing worth commenting on in all this is that we have
abandoned using cryostat sections whenever we can, since we have found
that for nervous tissue in particular, the freezing (even at -25 etc)
really damages the cell morphology. We no use polyethyliene glycol
embedding whenever we can - no shrinkage, no distortion, and the
sections remain fully hydrated throughout... (it's also in that Murphy
paper...)

Hope that helps - I'm sure there are more details that might matter, but
that's probably enough for now!

IAN




--
Professor Ian Gibbins
Anatomy & Histology
Flinders University of South Australia
GPO Box 2100, Adelaide, SA 5001
Australia

Phone:  +61-8-8204 5271
FAX:    +61-8-8277 0085
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