Immunofluorescence for nuclear antigen - Immunofluorescence (Mar/31/2008 )

I want to do Immunofluorescence for p63, which is a nuclear antigen....My doubts are (1) what fixative I should use to fix SiHa cell line for IF???? (2) Do I need to do permeablization as it is a nuclear Ag???? (3) wt reagents I should use for permeablization????

Thanks
Shweta

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if you dont know - experiment!

are these cells, frozen sections, wax sections

look in the protocols bit to give you some idea's

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Actually I am using SiHa cell line as a positive control for p63 expression....these cells are adherent to coverslips so I need not to go for any paraffin or frozen sections....for fixing these cells I am using methanol and Acetone (3:1)....for staining I will using Vectors lab reagents.....i will be blocking cells with the serum provided by vectastain than primary antibody incubatiuon at RT for 1 hr....washing....secondary ab incubation (biotinylated)....tertiary ab incubation (streptavidin FITC) conjugated....
I will be follwoing this protocol....will it be ok....

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eek!

you seem pretty sorted (although i use 1:1 methanol acetone and would use triton x for epitope retrieval (permeableisation) but then again i work on frozen skin - my techniques do cross over as i've tried em on cells too)

i take it your working from a kit (tertiary isnt an antibody) if you want to improve you need to know the animals the antibodies were raised in then block with serum of the secondary (biotin rabbit anti goat = rabbit serum)but you might be anyway and also block the avidin/biotin (also might already)

thing is i stick with my origional statement - if you dont know - experiment!

until the protocol makes sense to you and you can tweak it - you learn nothing

getting the result is only half of the experience

good luck

dom

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