Methods and Techniques Discussion => Apoptosis or Cell Death => Topic started by: Jossy on July 22, 2004, 08:26:22 PM

Title: help with Hoescht
Post by: Jossy on July 22, 2004, 08:26:22 PM
Hi, i want to see apoptotic nucleus in tissue sections of rat brain (criostate slices) and i need some advices with hoescht
I try in sections treated with H2O2 (in vivo injection in right septum), but i can't see difference between normal and damaged nucleus (it means, between rigth and left septum)
was the treatment with H2O2 too much?
I used Hoescht at 0,5 ug/ml, as the manufacturer sugest.
Title: help with Hoescht
Post by: ImmunoNYC on July 07, 2005, 12:50:00 AM
Why not immunostain for markers of apoptosis or do a TUNEL assay instead as what you are trying to do is very difficult.
Title: help with Hoescht
Post by: Jossy on July 07, 2005, 08:43:34 AM
I just did that injection as a control for the technique (I'd want to be sure that the staining was specific)
I can't make a TUNEL assay, because my slices are too thick (I did a try, but doesn't work)
But I already did some apoptotic markers (caspase 3, p53, etc) and my work it's finished.
Thanks
Title: help with Hoescht
Post by: ImmunoNYC on July 07, 2005, 10:25:14 AM
Well I am glad you figured everything out and your work is complete. I am wondering how thick were your sections that you could not do TUNEL?

Thanks!
Title: help with Hoescht
Post by: Jossy on July 07, 2005, 12:55:50 PM
I was using 30 micrometer thick slices of rat brain
there is a kit (I'm not working in that anymore and I don't remember the name) that works very good with that kind of slices, but in my lab, they already bought another kit (for cells) that just works with 6 micrometer slices.
Thanks for your ineterest
Title: help with Hoescht
Post by: excalibur on July 09, 2005, 11:38:13 AM
AHHHHHHHHHHH! Bad, Bad, Bad.

You Slice meat, histologists SECTION tissue!!!!!!!!
Title: help with Hoescht
Post by: ImmunoNYC on July 10, 2005, 02:39:45 AM
Hmm, I think most kits would work with 30 um tissue. It's not that thick. You might need a confocal for resolution of the staining but I dare say it would work as long as your permeabilized your tissue well enough. I could be wrong though, just my opinion.