My name is Ryan McTaggart.

I am attempting to use IHC to I.D. liver cells. hepatocytes, Kuppfer cells,
cholangiocytes, stellate cells, etc.

I am starting with Santa Cruz Biotechs (SCB) 
goat asialoglycoprotein antibosy and the 
goat desmin antibody

my secondary is donkey anti-goat conjugated to Texas red.

my protocol is not working

Questions:

SCB reports using the antibody in fixed tissue with antigen retrieval
I am using frozen with fixation in methanol shouldn't it work?

how important is it to use donkey serum in blocking buffer - can i use horse
- is it the same? I don't need goat serum right?

how soon do I need to freez the liver after it is removed from the mouse?
can I let it dehydrate in sucrose for several hours?

does anyone think antibody staining on a flat, non-agitating surface, with a
PAP pen is less effective than in a coplin jar with agitation?

finally, has anyone successfully used these antibodies and are the other
antibodies you have used successfuly on frozen liver sections to
specifically I.D. cells?

thank you for your time - i am a young researcher and your valuable
experience means a great deal to me.


best,

ryan mctaggart
UCSF - Transplant Research Laboratory