Methods and Techniques Discussion => Antigen Retrieval => Topic started by: Ghuman on October 19, 2003, 03:55:57 AM

Title: Citrate buffer antigen unmasking
Post by: Ghuman on October 19, 2003, 03:55:57 AM
Hi

I have used citrate buffer (sodium citrate- 1.47g/.5L; pH 6.0) for unmasking free floating 40 um sections (boiling on hot plate for 5 min). After unmasking the whole of the sections turn blue black (DAB) as compared to sections whcih are not boiled. Also, with fluorescent method, there is more non specific binding though there is increase in specific binding also for some antigens.
What acan be the reason for increase in non specific binding by boiling the sections?
Regards
Title: Citrate buffer antigen unmasking
Post by: ghostdog on October 20, 2003, 04:49:02 AM
Are you letting the slides cool down after the boiling , this is a big cause of non-specific binding of DAB
Title: Citrate buffer antigen unmasking
Post by: Ghuman on October 20, 2003, 04:55:34 AM
I boil free floating sections and let them cool for 3 min and then add cool citrate buffer to decrease the temperature quickly. what should i do is this cooling down ok .
Title: Citrate buffer antigen unmasking
Post by: ghostdog on October 21, 2003, 05:22:53 AM
yeah it sounds ok, have a look at the topic section on antigen retrieval
Title: try tris/edta ph9.0
Post by: cheewee on November 06, 2003, 11:58:04 PM
I use tris/EDTA, ph9.0 it's give me a better results, why don try?