Products and Equipments Discussion => Histology Products => Topic started by: qtian on July 06, 2003, 09:52:17 AM

Title: In Situ Cell Death Detection Kit from Roche
Post by: qtian on July 06, 2003, 09:52:17 AM
Hello,

Has anyone been using In Situ Cell Death Detection Kit (Fluorescein) from Roche (Cat#1684795) for paraffin sections successfully?

Thank you
Title: In Situ Cell Death Detection Kit from Roche
Post by: brian on July 06, 2003, 10:16:14 AM
Yes, I have used this kit and it works very well. Just follow the protocol provided with the kit. I also diluted 1:2 of the TUNEL mixture and it worked fine too.

Brian
Title: In Situ Cell Death Detection Kit from Roche
Post by: qtian on July 06, 2003, 11:37:02 AM
Thank you Brian. I will give it a try.
Title: Background (TUNEL)
Post by: annlouberg on August 27, 2003, 09:11:16 AM
I am using the TUNEL-test on paraffin embedded slides of eyes, but I have some problems with backgroud staining. My untreated controls also show quite a lot of staining (although the negative control without the TUNEL-enzyme is totally unstained). I use the protease-pretreatment step as recommended - could this increase the background (of course, I use nuclease-free protease)? Or should I just develop in DAB for a shorter period (now I use 8 minutes).
Title: In Situ Cell Death Detection Kit from Roche
Post by: brian on August 27, 2003, 08:10:49 PM
It is very likely that the protease pretreatment brought up the unspecific staining. You can use citrate buffer pH6 heating method which I found is superior than enzyme digistion.

Brian
Title: Thank you
Post by: annlouberg on August 28, 2003, 01:29:44 AM
Thank you for that, Brian. I will try it.

Kindly, Ann-Louise
Title: In Situ Cell Death Detection Kit from Roche
Post by: Scarr on April 13, 2005, 06:22:07 AM
Sorry to bump this topic, but I was looking for some advice with this kit.  I'm having difficulty getting a positive signal with my DNAseI control.  I don't think the DNAseI treatment is the problem itself, I believe the issue is with the fixation and permeabilization conditions I use.

I am performing my experiments on HeLa cells, using the conditions described in the handbook (2% Paraformaldehyde in PBS, 1 hour; followed by 0.1% Triton X-100 in 0.1% Na-citrate for 2 mins on ice), but have also tried 2% PFA in PBS, 1 hour; followed by 0.5% Triton X-100 in 0.1% Na-citrate for 10 mins at room temperature with no success.

Has anyone had successful experiments with this kit on HeLa cells?  I can obviously try lots of different conditions but was hoping for some time-saving advice!
Title: In Situ Cell Death Detection Kit from Roche
Post by: ImmunoNYC on June 17, 2005, 01:47:36 PM
Try to reduce your fixation time to 10 minutes.
Title: Re: In Situ Cell Death Detection Kit from Roche
Post by: Charming505 on April 15, 2009, 09:18:38 PM
hi...i've used this kit on paraffin embedded bone sections to test out if it works, but the signal is very week. Did anyone further amplify it with anti-FITC antibody & DAB?? if it works, what did people dilute the anti-FITC antibody in?? in PBS? Sorry..i have very weak background knowledge in in-situ hybridisation :-[.