Methods and Techniques Discussion => Apoptosis or Cell Death => Topic started by: gussy on September 05, 2010, 09:45:40 PM

Title: Antigen retrieval for TUNEL assay
Post by: gussy on September 05, 2010, 09:45:40 PM
Hi all,

Can I use Heat Induce antigen retrieval methods such as using citrate buffer instead of Proteinase K methods in TUNEL assay. My TUNEL kit manual using Proteinase K methods, and I need to buy proteinase K if I follow that manual

Thanks in advance

Title: Re: Antigen retrieval for TUNEL assay
Post by: richard03 on September 06, 2010, 12:10:04 PM
In my experience, the citrate buffer also works for TUNEL staining, but proteinase K seems perform better.
Title: Re: Antigen retrieval for TUNEL assay
Post by: gussy on January 23, 2011, 08:21:46 PM
Thank you for your kind advice, I have tried and it worked. Now I am performing TUNEL assay on cell culture, I have problem with methyl green counter staining, after 30 seconds of counter staining the stain was very dark, I think my TUNEL is okay because I can see some apoptosis cells, how can I reduce the counter staining color and save my slides?

Any advice will be very appreciate

Regards

Gus
Title: Re: Antigen retrieval for TUNEL assay
Post by: richard03 on January 25, 2011, 12:00:42 PM
Alcohol will remove methyl green quickly so you may want to rinse quickly in 95% alcohol after staining. Refer to this protocol.

http://www.ihcworld.com/_protocols/counterstain_solutions/methyl_green.htm