Methods and Techniques Discussion => Antigen Retrieval => Topic started by: cincanada on April 02, 2009, 11:08:57 AM

Title: Antigen retrieval
Post by: cincanada on April 02, 2009, 11:08:57 AM
Good Day, I have been working with Pannexin in immunohistochemistry and have even tried the antigen retrieval protocol (Antigen Retrieval Method for Free Floating Sections) on cryostat sliced sections at 30 microns.  It is there but the signal is very weak.  Is there another protocol I can try or change ........ anything would be greatly appreciated.

C

 
Title: Re: Antigen retrieval
Post by: cincanada on April 02, 2009, 12:58:37 PM
Good Day, I have been working with Pannexin in immunohistochemistry and have even tried the antigen retrieval protocol (Antigen Retrieval Method for Free Floating Sections) on cryostat sliced sections at 30 microns.  It is there but the signal is very weak.  Is there another protocol I can try or change ........ anything would be greatly appreciated.

C
Title: Re: Antigen retrieval
Post by: richard03 on April 02, 2009, 11:08:42 PM
Did you use Triton X-100? How long did you incubate the primary and secondary abs?
Title: Re: Antigen retrieval
Post by: MT Scientist on April 06, 2009, 03:16:21 PM
Good Day, I have been working with Pannexin in immunohistochemistry and have even tried the antigen retrieval protocol (Antigen Retrieval Method for Free Floating Sections) on cryostat sliced sections at 30 microns.  It is there but the signal is very weak.  Is there another protocol I can try or change ........ anything would be greatly appreciated.
C

Was your tissue fixed or fresh frozen?  If fresh frozen, you may wish to try without the antigen retrieval, as it may be unnecessary.  Try changing the concentration of primary and/or secondary antibody or try an amplification method.
Title: Re: Antigen retrieval
Post by: cincanada on April 07, 2009, 11:14:13 AM
Tissue is fixed in PFA, sliced at 30 microns on a cryostat and free floated.  Blocking agent is 1% BSA, 0.3% Triton X, 2% Donkey Serum. Incubated in 1Ab 24 hours, 2Ab 2 - 24 hours. Using maximum recommended concentration.  I have tried it without the antigen retrieval, this is my next step and it has not been that successful with the one sanitgen retrieval method I have tried (used 2% Nonfat milk, 0.3% Triton X, 0.1% Sodium Azide in PBS, off of ImmunoWorld web site).