[Histonet] immuno fluorescent stains on frozen rodent tissues


I was hoping to find another researcher who is doing immuno fluorescent
stains on frozen rat tissues. Especially, anyone who is using an
Autostainer to stain these slides. I am attempting to stain with both CD3
and CD8a (biotinylated antibodies) on frozen spleen tissue. I can get both
stain protocols to work but once I put the 2 of the protocols back-to-back
on the Autostainer, the tissues want to either fold over or completely wash
off the slides. I am using a good quality charged slides, cut 7 micron
sections, allow to dry, fix with acetone, dry again and then store in the
-80C freezer until the next day. I then remove the slides, allow to dry
again, fix again with cold acetone, hydrate quickly with buffer, then begin
my stain run, I have the Dako autostainer and have removing all forceful
"wash buffer" steps with simply buffer dropped on as a reagent, and then
blown off. The tissues have held on through the first primary antibody but
can't make it to through the second primary protocol. Any suggestions would
be very welcome. The only alternative will be to go back to manual stains
and this group I'm working this up for has many, many tissues to stain.

Warmest regards,

Terri A. Prestegard
3M Center, 270-3S-05
3M Pharmaceuticals
St. Paul, MN 55144-1000
Histology Archivist
Senior Histotechnologist
Tel: (651)-736-7519
Fax: (651)-737-5506

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