I tried to do ZO-1 (1:50) staining in C57bl/6 retina flatmount. and as secondry antibody I used goat anti rabbit IgG conjugated alexa fleur 488 (1:500) also for blocking I used 0.1% triton X-100, 5% BSA in PBS for 1 hr at RT. But as you can see in attachment I see all the background green. I try to solve it if you have any experience by ZO-1 staining in flatmount could you help me.
I would also recommend that you use a full dilution series, including both a negative and a positive control. I would always match my serum to the host of my secondary, in this case I would use goat serum instead of BSA. This should help to reduce the non-specific binding of your secondary antibody. You can use anywhere from 3-10%.
Hi Saeideh,
How did you processed the samples before fixing? Where the retinas kept in cold buffer for too long?
Cheers,
hi Sebastian
after enucleation i wash the eyes twice by PBS , punch the cornea by needle and fix in PFA 4% for 2hrs in 4C then wash them 2 times with PBS and keep them on PBS in 4C until doing blocking and staining.
Dear Saeideh, I asked about the pre-fixation temperature because tight junctions are structures highly dependent on the integrity of the actin citoskeleton and therefore sensitive to low temperatures. At low temperatures actin filaments tend to depolymerize, consequently proteins like ZO-1 may become cytosolic by losing its association with the actin network or because are not longer properly sort and delivered to tight junctions. My guess is that fixation at 4C doesn't occurs fast enough and may perturbs the actin cytoskeleton causing the delocalization of ZO-1 from the tight junctions into the cytosol.
Perhaps, if your protocol allows it, you can try fixing less time but at room temperature or anywhere between 30-37 C. Then you can quench the aldehydes with PBS-Glycine 0.1M for 15-30 minutes.
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