[Histonet] NonSpecific Staining Throughout Frozen Tissue

[Andrea Kwong]

I am using an anti-Sox9 rabbit polyclonal antibody to stain frozen
OCT-embedded porcine heart valve tissue, but am getting non-specific
staining throughout my sections. I do not think it is something to do
directly with the primary antibody because I have been getting this
nonspecific staining even on sections with no primary and IgG control and
with another antibody (anti-SMA) that we have previously used successfully
on frozen porcine tissue. My protocol is as follows:

Briefly:
1) Warm slides at room temperature for 20 min
 2) Fix in acetone 10 min
 3) 3x3min PBS/Tween Wash
 4) Peroxidase Block 10 min (3% H2O2 in Methanol)
 5) 2x3min PBS/Tween Wash
 6) 45 min Horse Serum Block (Vectastain Elite Kit)
 7) 1 hour Primary Antibody
 8) 4x3min PBS/Tween Wash
 9) 30 min Secondary Antibody (Vectastain Elite Kit)
 10) 4x3min PBS/Tween Wash
 11) 30 min Vectastain ABC (Vectastain Elite Kit)
 12) 2x3min PBS/Tween Wash
 13) 5 min Vector Nova Red
 14) 5 min dH2O
 15) Hematoxylin Counterstain
 16) Graded alcohol dehydration to xylene
 17) Mount with Krystalon

I previously thought it may be an issue with endogenous biotin, but I did a
run without serum block, primary and secondary... but there was no
staining. I'm thinking it may be an issue with fixation affecting
secondary, but I'm not sure what to change and was hoping for a second
opinion.

Thanks for the help