In my experience the longer you incubate the stronger the signal. So no harm if you incubate too long. Just make sure that you wash carefully with at least three changes of buffer.

I guess it depends whether you have had problems with background before. But, generally, it's fine to leave the secondary for a bit longer in case you blocked your sections. And yes, make sure to wash properly. Good luck!

I agree if background is not a problem, incubating longer with secondary is not a problem. If you have background longer may increase the background and so will have to wash longer.

I agree with the answers above.  However, I think that I would err on the side of leaving the primary longer at 40C.  Just my personal preference.  I have primary antibodies that I incubate for 3 days at 40C, but they are also titered for that incubation length.

For most antibodies of the correct dilution (no background), 2 hours RT incubation is equivalent to overnight at 40C.  So, if worst comes to worst, incubate the secondary overnight at 40C also.  I have done this due to unforeseen circumstances without an increase in background.  I have also done this with ABC reagent without problems.

Good Luck!

Jill

Hello,

I agree with Jill and Richard

Leaving the primary longer at 4C is usually OK unless you have non-specific staining, but the secondary can cause problems.

I agree  with Jill, Richard and Ariel too. My experience is: you can stopp your immuno  in the buffer for any time. I had to leave sections  after the primary ab incubation in PBS for a week. When I finished  my immuno with secondary ab , ABC and DAB-reaction I didn't  find any diferences between this and  privious results. To know this will help you to make your immuno organisation more comfortable.

leaving membranes in primary antibodies for longer time is always better than leaving them in secondary antibodies. I regularly leave membranes in primary antibody for the weekend and finish it off on Monday with secondary for 1 hour at room temperature.

I would do a longer incubation with primary antibodies rather than with secondaries. If you perform 1AB incubation at lower temp (e.g. 4 degree celsius), it may actually slow AB bindings and reduced unspecific bindings. Longer secondary antibodies may have generate higher background signals.

i'll opt for the first option and i've already done it wthout any problem.with the second option, i think you risk having a lot of backround noise

I have primary antibodies I incubate even for 48 hours at 4C (Brain slides/30 to 40 um thick). I always avoid long times in the secondary antibody due to background.

Hi

I am doing immunostaining for Dengue virus. In which I give incubation of primary & secondary antibody for 1 hour. I am getting good results, but my assay is cell culture base.