4 replies to this topic

[The aussie lab]

Hi guys,
I am new to immunofluorescence and I am about to try to visualize a protein in my bone marrow derived macrophages. The problem is that the secondary antibody Alexa fluor that i ordered has been delayed and I have my cells ready on my coverslips but not all the reagents. My question is can I start the IF procedure and stop somewhere before the 2ndary?? I know that for tissue you are able to leave them in Ethanol for a while after PFA fixation but  I have no idea for cells... Thanks a lot for your help, LN

[bob1]

You can definitely stop them post fixation.  Store as you would for tissue in ethanol.

[fraffly]

I'd store them in PBS. We usually just pipetted the PFA off after fixation and added PBS, then there is also a rest of PFA present. This way the cells stay quite stable for a week or two, we never checked longer than that.
Don't forget to quench free aldehydes before labeling, they can bind to the primary Antibody and give false positives, we used
50mM Ammonium chloride in PBS for 5-10min on 4°C.

[bob1]

To add to Fraffly's advice, storage in PBS will only work for a couple of weeks max.  After this the cross-linking degrades so staining often looks funny.  Ethanol storage is better for long term, but they will need to be re-hydrated before staining.

[The aussie lab]

_{Great, thanks so much for your help!}