Toto-3 staining - (Feb/15/2007 )
Hallo, I'm trying to stain DNA with Toto-3 in whole mount preparations of Drosophila larval brain neuroblasts. Somehow it doesn't work and I don't know why. I used 2 micromolar concentration of Toto-3 in PBS a stained it for 1 hour at RT. I heard that Toto-3 is sensitive to pHb but I didn't find at what pH it works. Could somebody help me? Thanks
-Lucia M-
i tend to use toto on parrafin or frozen sections and its just a case of leaving it on for twenty minutes and its pretty foolproof - the ph of the sample is buffered by the use of the pbs as a wash.
it sounds to me more like a question of absorbtion - do you pretreat the tissue in any way which would aid the toto getting into the cells and then the nucleus
-Dominic-
QUOTE (Dominic @ Feb 16 2007, 02:15 AM)
i tend to use toto on parrafin or frozen sections and its just a case of leaving it on for twenty minutes and its pretty foolproof - the ph of the sample is buffered by the use of the pbs as a wash.
it sounds to me more like a question of absorbtion - do you pretreat the tissue in any way which would aid the toto getting into the cells and then the nucleus
it sounds to me more like a question of absorbtion - do you pretreat the tissue in any way which would aid the toto getting into the cells and then the nucleus
I used Toto-3 in whole brains and I'm using simple protocol. I used to permeabilze tissue in PBS+0.3% Triton for 10 or 15 min and the next step are performed in PBS+0.1% Triton. My boss told me, that it doesn't work probably due to pH. But I didn't find what should be the pH of solutins.
-Lucia M-