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I just finished an immunofluorescence experiment and I'm wondering what went wrong. The tissues seem dimmer than they should be.

One mistake I made was: I completed the antigen retrieval step, in a 90 degree sodium citrate bath for an hour. The next step is to cool the tissue in ice for 8 minutes before washing. I forgot to cool the tissue, and just put it straight in the wash.

Could this rapid temperature change have affected my results? How important is this step?

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  • $\begingroup$ Are you using secondary immunofluorescence? If you are, the 8 minutes of cooling are probably intended for the signal amplification. In other words, as you leave it for the time specified, the flourochromated secondary antibodies have more time to attach themselves in greater numbers to the primary antibody without your sample disintegrating. $\endgroup$
    – rotaredom
    Jun 27, 2016 at 20:21
  • $\begingroup$ I do use a primary and a secondary. But the cooling step is before any antibodies are added. Rather, it goes: deparaffinization, antigen retrieval (90 degrees citrate buffer), 8 MINUTES ICE, pbs wash, blocking, primary antibody. Honestly, I'm not sure what the cooling step is for. I was thinking it was just to prevent rapid cooling of the glass slide, so it doesn't break. But I'm hoping to know if it has anything to do with the tissue. $\endgroup$
    – amd1972
    Jun 27, 2016 at 20:44

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