help in immunocytochemistry - (Jul/10/2007 )

I never do blocking, per se. For normal IF, I incubate fixed cells with Abs diluted in FDB (buffer includes BSA and FBS and NGS). For live cells, I incubate them with Ab in normal growth medium, but I let them internalized the Ab after that in most cases, though, so I guess that incubate live cells with Abs in FDB is also fine. Or else you may want to block before Ab incubation. Maybe someone has done this before can help?

Hi! Minnee,
I still feel that you should not bother about some cells that show internal staining even if you do not permeabilized them. But only if you feel that the majority of them show only surface staining. I would suggest that you first fix with 3% PFA (do not leave longer, 10 min should be sufficient), Block, and then incubate with primary and secondary Ab (preferably in 1%BSA in PBS).

Good luck

Hello there!
I am also new to immunofluorescence pondering over similar questions. Like minnee I do NOT want to permeabilize my cells (HEK293). I wonder whether fixation with paraformaldehyde could be a problem here and (provided that yes) whether it is necessary at all. Is it possible to stain without fixation? What could be a disadvantage if I leave it away? What is it good for anyway? I know it crosslinks proteins, but I do not see how this translates into a better staining. Almasy suggested to first incubate with the 1st Ab, then fix and then apply the 2nd Ab. In my case the 1st Ab is already labeled so there is no 2nd Ab. Should I nevertheless fix after staining? And one more question concerning fixation: paraformaldehyde works on free amino groups (right?). My protein to be detected has a FLAG tag in an extracellular loop. The FLAG sequence is DYKDDDDK so there are 2 Lys with free amino groups that could be targeted by paraformaldehyde. I worry this could prevent Ab binding to the tag. Any suggestions? Thx!

I tried to do staining on ice.But all the cells were dead.
Can anyone please help me how to stain proteins on the membrane only?

What did you do? How long did you incubate on ice? What cell is this? How do you know that cells dead? They washed off after first incubation?

I incubated normal human fibroblasts with primary antibody for 1 hr on ice.After that i checked under microscope and most of the cells were washed off but few were still there.After incubation with secondary Ab,all cells were lifted up.
I also checked the cells after washing with PBS & before incubating with pri.Ab,they were fine.

No,i didn't incubate cells with FDB diluted Ab.I diluted Ab in 2% BSA.I think u r right that if cells were washed off after first incubation.
Can u please tell me why should i use FDB & give me the recpie for it?I think that may be a problem.
Thanks a lot for ur help.

2%BSA in PBS or H20? The reason why I asked how did you incubate your live cells with Abs is because if you just incubate them with Abs in, say, H20, then cells will die. When I do my antibody/ligand internalization assay, I also have to incubate live cells with Abs or ligands and then fix for surface binding. For such exps, I incubate cells with Abs/ligands diluted in normal growth medium (ice-cold though, and chilled the cells before incubation, all on ice). The FBS in growth medium should help for blocking purpose, and I think that cells may be happier. FDB is used for blocking and diluting Abs, same as 2%BSA in your case, but it may block better. Formula: 2%BSA, 5%FBS, 1mM MgCl2, 1mM CaCl2 in PBS. Sometimes I add 5%NGS also, but not always. After the incubation (cells (on coverslips) facing down on the LID (aka the cover) of the plate, DO NOT incubate INSIDE the wells where you growing cells), put the coverslips back inside the wells for washing, cells facing up. Rinse gently with cold PBSCM a few time, briefly, then fix (with cold PFA). Then you can do whatever you want normally.