I'm just about at wits end with this protocol -- any help would be much appreciated.
Here's the story -
Tissue = Neonatal (PND7) Rat
Perfused, 4% phosphate buffered formaldehyde
Post-fixed 24hs in 4% phosphate buffered formaldehyde
Sunk in sucrose (30%)
Flash frozen in isopentane and stored at -80C until cryosectioning
Sectioned at 20um, -15C, stored at -20 until processing.
Air dried 30' then 30' at 50C
ApopTag (Chemicon) and TACS TUNEL (RnD Systems) kits used.
Antigen Retrival --> Tried Proteinase K (1:50 and 1:200) at RT, Citrate Buffer (boiling), Cytonin, Tween, Triton
TdT --> 30mins, 45mins, 60, 90, 120 (RT and 37C)
Detect via anti-digoxigenin HRP (in the Chemicon kit) or stepavidin-HRP (RnD) or ABC (Vector) and DAB
Positive controls generated following Ag retrival step, via treatment with DNAse
We get great staining (as expected) with the positive control slides, but zero in the non-DNAse treated slides.
I know that these sections have dying cells, the animals from which they came (and this is across several batches of animals) were treated in a manner that has given our lab lots of positive results before.
The change from previous runs is the use of fixed tissue...
Any suggestions would be very greatfully received.
-- P.A. Forcelli, Gale Lab, Georgetown University