Immunocytochemistry showing high background - (Mar/31/2008 )
I am doing suspension culture for limbal stem cells. These cells are not adherent so i am taking them on poly lysin coated slide and then fixed with methanol or acetone....But I am facing problem with ICC...Getting high background whatever antibody i am using its giving totally dense brwon stain even in negative control also....what could be the reason....n what could be the avenues....????
Are you staining before or after fixation?
if you are staining extracellular antigens you could maybe stain before fixation?
maybe try to an other fixation permeabilization procedure, like PFA and saponin or triton. Some antibodies prefer methanol, some other prefer PFA...
if you are staining extracellular antigens you could maybe stain before fixation?
maybe try to an other fixation permeabilization procedure, like PFA and saponin or triton. Some antibodies prefer methanol, some other prefer PFA...
First I fix my cells with methanol and acetone then I do staining....Do i need to do permeabilization as i am only looking for surface markers.....????? i.e. cytokeratin 3.....I am using Novacastra kit (IHC).....Even in antibody catalouge they have not prefered any method for fixing cells....
No, you don't need to permeabilize for surface markers.
I know that some antibodies don't like PFA, and then it is better to stain before fixation. I don't know about methanol.
someone told me that depending on antibodies (and you only know by trying) the staining will be better if you fix with methanol or if you fix with PFA.