immunocytochemistry troubleshooting - (Feb/23/2007 )

HI all
I am new to immunocytochemistry. My slides are not of good quality. The fluorescence is very diffused. I use triton 0.1 triton to solubilize the cell membranes (my proteins of interest are intracellular) and use my secondary antibodies at 1/1000 concentration. I really need some general tips and if possible a protocol for slides that were good (even if I know that every case is different)
THanks

-kalfeb-

QUOTE (kalfeb @ Feb 23 2007, 09:30 PM)

if you work at CLSM, voxel-number may be too low; use Nyquist theorem to calculate;

other problems may arise from not well working primary antibodies because inappropriate to ICC or too diluted ( often range between 1:50 and 1:100); for secondary, use Alexafluor-labeld Ab´s; good results in ICC depend much on trial and error using different methods and antibodies;

-The Bearer-

for the secondaries 1:300 works with most alexa fluors(invitrogen - mol probes) and biotinylated this anti that (vector)
this leaves you free to try a simply assay testing your primary concentration - i have antibodies for two isoforms one is 1/50 the other is 1/2000.
if the diffuse staining is a bit halo like it may be you are visualising too late after staining.
you can always fall back on a middle avidin/biotin step if its just a lack of signal.
also what do you fix your slides with - a cross linker like formaldehyde may need something more potent that triton x (0.2%) - or do you use a methanol:acetone type in which your ok

-Dominic-

perhaps your diffuse fluorescence is auto-fluorescence - you may want to include a quenching step prior to blocking and post permeabilising -50 mM ammonium chloride (in PBS) for 10 min
also, maybe increase your blocking time?

-aussieuk-

Thank you all for your suggestions. I will do again my immuno and update the results.

-kalfeb-