I am working with a breast cancer cell line which has a high autofluoresence. Does anybody can give me a clue to reduce autofloresence?
I used non fixed cells for incubation with primary antibody then cells were washed few times and fixed with PFA 4% washed twice and incubated 1h with secondary antibody Alexa647, washed few times stained with DAPI for 15min, washed and added antifade diamond from invitrogen, then assessed.
I would recommand to fix the cells first before you start your immuno. Permiabilisation in 0,2 - 0,5% Triton-X containing PBS or TBS before the blocking step will help the primary ab to get into the cells.
Sometimes PFA increases the autofluorescence. In that case try to fix your cells with aceton. Negative controls (immuno without primary ab, if it is possible) will help to destinguish autofluorescence, unspecific background staining from the real lable of the primary ab.
Try to use less primary and secondory antibodies. I think this will reduces the background.
Hi, I agree you should try fixing before your 1ary if you can. I suspect that fixing after incubation increases your non specific binding by stabilizing weak bonds.
I'm surprised you have autofluorescence in the 647nm channel, we usually have to deal with it in the green and/or red channels only..
thanks for all your response. The simmunotaining does not work if I fix cells first apparently it interfer with epitope detection and also I tried TX-100 but with saponin for detection of membranar protein works good for other cell lines with the same antibody.
I recommend we back up and test the hypothesis that it's actual autofluorescence (vs background from nonspecific binding of secondary or nonspecific signal from nonspecific binding of the primary).
If it is autofluorescence, it will exist in the absence of the immunostain. Try imaging naive cells, and compare to cells after different periods and methods of fixation (for starters, try 1 or 2 % PFA, or cold acetone, methanol or ethanol).
Image in as many channels as you have access to, characterizing the autofluorescence helps pinpoint its source.
If these controls are all negative, you likely have nonspecific Ab binding. Check for problems with the 2ndary by running No-primary controls as Ute suggested from the start..
If it is indeed just true autofluorescence, first consider switching to channels where it's less of an issue. Beyond that, you can try chemical quenching (no idea how or if that is done for cells, I work on tissue..), or, carefully, digital compensation after imaging the cells before secondary incubation ...
If it is nonspecific staining, you should first try to reduce concentrations or incubation times, and see if you can keep high affinity specific binding while reducing background like that. Another thing to consider is changing your wash solutions to include different block.
All that being said, I do tend to agree with Hai, I'm skeptical that you have biological autofluorescence in the 647 channel
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