DAPI staining

Hi all,

I have a 2mm thick slice of rat brain, and I'm DAPI-staining it at a 10nM concentration at the moment (because thats the number I kept stumbling across). I've left it for roughly a week now, but I'm unsure how long I should leave it. It appears to vary a lot with staining type.

How long should I leave it, and how long should I wash it? Has anyone tried DAPI?

When I stained for perineuronal nets, I got a lot of unspecific binding on the very surface (not too much background other than that though). Would that be due to insufficient washing perhaps?

Cheers.

Comments

  • lijunlijun Posts: 14
    May be you can try to label the section for 24h with Hoechst(1ug/ml) and don't wash. or you can even add Hoechst in the RI matching solution(eg.FocusClear).
  • AdamAdam Posts: 31
    I've used DAPI a few times in whole and sectioned mouse brains. I've had full penetration of a mouse brain after 24 hours incubation at room temp. I would say a week is certainly enough, DAPI is orders of magnitude smaller than antibodies etc.
  • BJ2014BJ2014 Posts: 8
    Usually 24 hours at room temp is sufficient to penetrate upwards of 3-4mm thick tissue. I use 1:1000 concentration.
  • What concentration [mg/ml] is used for immunohistochemistry with DAPI in a whole brain clarified PACT? Did it work well? The product number and trading house? Thanks!
  • I did Hoechst staining, 1:500 in PBS-T overnight, then wash 3 times in PBS-T for ~ 2 days - ready to image.
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