Tina Carvalho
Aldehyde fixation, even with gluteraldehyde, should not prevent
lectin binding. We've had good success with a wide range of lectins
on formaldehyde and glut. fixed human material, both LR white and
araldite embedded (we prefer LR white). To visualise bound lectin, we
used biotinylated lectin followed by gold labelled antibiotin. A
detailed method is given in:
Localisation of alpha(2,3) and alpha(2,6) linked terminal sialic acid
groups in human trabecular meshwork. SA Chapman, RE Bonshek, RW
Stodart, KR Mackenzie, D McLeod. British Journal of Ophthalmology,
1994; 78:632-637.
We modified a method described by Slot and Geuze:
JW Slot and HJ Geuze. In: Polak JM and Varndell IM, eds.
Immunolabelling for electron microscopy. Amsterdam: Elsevier
Scientific. 1984: 129-142.
We've also visualised lectin binding (including ConA and WGA) in
resin embedded tisue at the LM level:
Glycoconjugates of the human trabecular meshwork: a lectin
histochemical study. SA Chapman, RE Bonshek, RW Stoddart, CJP Jones,
KR Mackenzie, E O'Donoghue, D McLeod. Histochemical Journal, 1995;
27:869-881.
This gives specificities, controls, etc, for a wide range of lectins.
Richard Edward Bonshek
No problems with aldehydes, they should not interfere with lectin labeling.
Should you wish to combine glycoconjugate detection with immunocytochemistry
at a later stage of the project, I'd go easy on glutaraldehyde: 0.1% max,
which should be enough to preserve the Golgi apparatus fine structure. In
addition, I'd suggest to limit the length of your fixation to 1 - 2 hrs.
After washes you can always store the material in buffer with azide at 4 C
for a while. (Please don't ask "How long is a while?", I did not investigate
the upper limit but had no problem with tissue and cells kept for 2-4 weeks
before processing. Of course, the sooner embedded, the better, as goes the
Bio EM CW).
Blocking agent: I always include a solution of 50 mM ammonium chloride in
buffer as 3rd and 4th washes (15 min ea.) after fixation to block free
aldehydes.
Embedding: I used Lowicryl when doing lectins and the results were great. I
suppose the other usual metacrylates should be fine as well.
A start is direct lectin-gold, quick and easy. You have the option to add
amplification by using biotinylated lectins and streptavidin gold. There are
lots of variation on the technique in terms of probes, detection methods and
so on.
The possibilities are, well, quite endless.
For info on lectin gold methods, the key authors to look for in your
reference database are Juergen Roth or Moise Bendayan in the 80's.
I hope this helps (and gets to the middle of the Pacific in time!)
Michel Deschuyteneer