RE: [Histonet] IHC on frozens

From:Guillermo Palao

Hi Sarka,
  I have recently had similar problems with parafin vs frozen sections, and these are the conclusions I have come to:
  - We routinely store at -20 ºC fridge the frozen sections already fixed for 5 min in acetone at -20 ºC, I do not know if this is the best way to do it, and I would appreciate any more informed input in this sense.
  - For quenching endogenous peroxidase you may use H2O2 0.3% (instead of 3%) for 5 min RT (instead of 30 min).
  - In my (little) experience, Ab concentrations do not matter that much if the antibody is good. For convenience purposes I stain frozen sections for 1 hour RT as compared with O/N at 4 ºC for FFPE sections.
  - We noticed similar (if not worse) background problems due to endogenous mouse IgG in frozen sections, although I did not do a side by side comparison.
  I found a really good improvement in terms of background after blocking endogenous biotin with Vector Avidin/Biotin blocking kit. Try staining your slides only with ABC reagent (no Abs at all), and if you see color developing (as was my case) then you definitely need to block endogenous biotin.
  Good luck,

Sarka Lhotak  escribió:
  Hello Netters,

I am trying to get some antibodies work on mouse tissue (see my
previous posting). Since I have not been successful on paraffin
sections I now turned to frozens where I have no experience. I am
getting the same staining (or background of course) on positive and
negative slides (antibody omitted). Even antibodies that I know work
very well on paraffin (my positive control), give me no specific
staining and a background mainly on connective tissue. I've searched
for a proper fixing, drying, dipping in water etc. procedures prior to
staining. I found it very confusing, people swear by different, even
opposing protocols.
So I have a few questions:
What is the best way to prepare/store your sections prior to staining?
Is quenching peroxidase and blocking the same as on paraffin?
Generally, are the primary and secondary antibody concentrations the
same as on paraffin?
Is the MOM problem worse on frozens than on paraffin?

All suggestions are welcome.


Sarka Lhotak

McMaster University
Hamilton, Ontario 

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