🚀 go-pugleaf

We have been labelling whole mounts and thick sections for many years for
multi-colour immunofluorescence. Several things seem to matter, but there
are also many ways of solving the problems... The fixative matters quite a
lot - buffered formaldehyde is fine, but we generally use so-called
Zamboni's fixative which is 2% formaldehyde and about 0.2% picric acid in
phosphate buffer. The picric acid seems to do something to the cytoplasm
that aids antibody penetration (the chemistry of picric acid is largely
unknown, I think), but it does not affect the immunoreactivity of most of
the peptides, enzymes or structural proteins we have localised over the
years. Glutaraldehyde is a big barrier to most antibodies. We need to use
it  for ultrastructural work, but at very low concentrations (eg 0.05 - 0.1%
) This will give pretty good ultrastructure when used with picric acid and
formaldehyde, but still allows good penetration.

After fixation, the tissue needs to be permeabilised with something. We've
tried and tested most things: detergents tend to damage the overall tissue
structure too much. For tissues containing large amounts of fat, the
dehdyration  to xylene and rehydration back to buffer works really well.
Often, though just a few washes in DMSO (dimethyl sulfoxide) is enough. In
some cases, tissue can be washed for a few minutes in glycerol.

We do not add anything to our antibody solutions, although some colleagues
swear by adding a small amount of triton to the mixture. Others always
incubate at 37 degrees, although we use room temperature without any
problem. Increasing the lenght of time for the incubations (eg 2-3 days)
really helps in many cases.

The embedding medium and method of cutting seems to make a difference:
cryostat sections give the best penetration, but worst structural
perservation. We now have methods for polyethyleneglycol and low-melting
point wax that wotk well for sections up to 40 um. For sections thicker
than that (up to 100um) we use a vibrotome. In these cases, we often
treat the section (not the precut tissue) with DMSO, ethanol or detergent
to aid penetration.

We also have found that there are large differences in the apparent
ability of different antibodies (primary or secondary) in penetrating
tissue. Soem get through easily, whilst other only label the surface of the
sections, no matter what you do to them...

Despite all this apparent success (!), there are some tissue which present
real barriers to antibody penetration. Many blood vessels are difficult to
use in whole mounts because the antibodies seem unable to penetrate through
the connective tissue matrix of the adventitia - they only get in via a cut
surface, so we are forced to cut the tissueinto thin strips to get good
labelling. I don't know how widespread this particular problem, but I'd be
intersted to know! (don't use enzymes to digest the matrix if you want to
preserve some of the any of the tissue strcture at all!!)

I hope some of that helps...
Ian Gibbins
Department of Anatomy and Histology
Flinders University of South Australia
Phone:  +61-8-2045271
FAX:    +61-8-2770085