DAPI staining

Tully

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.

lijun

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).

Adam

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.

BJ2014

Usually 24 hours at room temp is sufficient to penetrate upwards of 3-4mm thick tissue. I use 1:1000 concentration.

jose_jpr

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!

snarker

I did Hoechst staining, 1:500 in PBS-T overnight, then wash 3 times in PBS-T for ~ 2 days - ready to image.