I am trying to standerdize the protocol to evaluate the presence of
TNF-a in the 10% neutral buffered farmaline fixed (3-4 hr) 4 micron
liver sections of mice. My +ve controls are LPS -treated (1 mg/Kg i.p.
1hr before killing) liver slices. I am using PAP method. Breifly,
Sections are heated @60oC for 30' to liquify parafin, two changes of
Xyline for 5' each, hydrate, wash in Tris buffer (PH 7.6). After that
dip in 3% H2O2-wash - Proteinase K (25u/ml)@ 37oC for 30' - Wash. After
that Block with 10% goat serum for 30' - wash- Anti mouse TNF-a in mouse
(1:200 of 1 mg/ml) overnight @ refrigerator - wash - Link ab (anti
rabbit Ig-G in goat 1:200 of 1 mg/ml) for 30' - Wash - PAP (1:200 of
1mg/ml) for 30' - wash - DAB for 10' - Wash Counter stain - wash -
butanol - Xyline - Mount. All reagent are fresh and followed according
to the manufacturor.
RESULT= NO +VE STAIN IN ANY SLIDES INCLUDING CONTROLS IN 3 DIFFERENT
I appreciate any expert comment on this method and proper
modifications.Thanks for the patience and help.