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Author Topic: help with Hoescht  (Read 9636 times)

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Offline Jossy

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help with Hoescht
« 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.
ossy
jossy@neurobio.pitt.edu

help with Hoescht
« on: July 22, 2004, 08:26:22 PM »

Offline ImmunoNYC

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help with Hoescht
« Reply #1 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.

Offline Jossy

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help with Hoescht
« Reply #2 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
ossy
jossy@neurobio.pitt.edu

Offline ImmunoNYC

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help with Hoescht
« Reply #3 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!

Offline Jossy

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help with Hoescht
« Reply #4 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
ossy
jossy@neurobio.pitt.edu

Offline excalibur

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help with Hoescht
« Reply #5 on: July 09, 2005, 11:38:13 AM »
AHHHHHHHHHHH! Bad, Bad, Bad.

You Slice meat, histologists SECTION tissue!!!!!!!!
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

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help with Hoescht
« Reply #6 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.

help with Hoescht
« Reply #6 on: July 10, 2005, 02:39:45 AM »