Immunofluorescence background - (Jul/25/2007 )

Hi
I am getting an incredibly high background from my immunofluorescence when I use only the secondary antibody. Does anyone have any tips on how to reduce the non-specific binding? I am blocking with 5% normal serum and washing with PBS for 3 times (5 min each) between steps, should I change the blocking or increase washes? Both my primary and secondary antibodies are diluted in blocking solution. I also have a problem with auto-fluorescence when I check cells alone with no antibodies and reducing the wavelength spectrum didn't help.
Any info will be gladly accepted, thanks

-syaniv-

Using high dilution of secondary antibody can reduce bgd.

-genehunter-1-

you may end up needing to find a new secondary? or use a bit less -

the autoflourescence may be a problem and I don't have any tips for you there.

aside from a titration of secondary to find the best concentration, the only other thing I can think of off-hand is that perhaps you could try, as a control, and isotype-matched negative (non-binding control for your assay) antibody for your primary, to bind up non-specific sites?

-aimikins-

Are you working with fixed cells? What fixation method is it? If you are using PFA, make very sure that you had washed away all PFA after fixing, since this can cause high background.

-Almasy-

QUOTE (Almasy @ Jul 26 2007, 01:20 AM)

Are you working with fixed cells? What fixation method is it? If you are using PFA, make very sure that you had washed away all PFA after fixing, since this can cause high background.

I'm using 10% normal goat serum, block 2hr. background is acceptable. and you also need 2nd Ab titration.

-rabbit-

how long are you blocking for?? That might be the problem.

-labrat612-

QUOTE (labrat612 @ Jul 27 2007, 07:37 PM)

how long are you blocking for?? That might be the problem.

software programs to work with CLSM data have the opportunity to reduce autofluorecence; I think there is also a plugin-in for ImageJ available; massive autofluorescence however is not to solve by manipulartion of images

-The Bearer-

I've use several different fixation methods for cells, methanol:acetone/10% formalin/PFA. None of them produce much background in cells (tissue sections are more tricky) if a good secondary is used (ty the alexa secondarys from invirogen). To block i've use 10% serum or 5% BSA -both work just as good.

-SARESK-

Did you wash the cells before fixing them? media could cause autofluorescence.

-scolix-