Wanted! Protocol Immunocytochemistry with HRP-DAB reaction - Need Non-fluorescent immunostaining on cultured cells (Dec/13/2007 )
Hi all,
I plan to do some immunocytochemistry with cultured cells on the coverslip. I need to use HRP in the secondary antibody-DAB reaction instead of fluorescent dye-labeled secondary antibody. Can anyone post or upload good protocol for this purpose?
Thank you all in advance.
-cancerous-
QUOTE (cancerous @ Dec 13 2007, 09:34 PM)
Hi all,
I plan to do some immunocytochemistry with cultured cells on the coverslip. I need to use HRP in the secondary antibody-DAB reaction instead of fluorescent dye-labeled secondary antibody. Can anyone post or upload good protocol for this purpose?
Thank you all in advance.
I plan to do some immunocytochemistry with cultured cells on the coverslip. I need to use HRP in the secondary antibody-DAB reaction instead of fluorescent dye-labeled secondary antibody. Can anyone post or upload good protocol for this purpose?
Thank you all in advance.
After the incubation with yoyr secondary antibody, you have to wash it 3 times (TBS or PBS). Then you make a solution of 5ml TBS (or PBS), add 4 drops DAB and 2 drops H2o2, you incubate the slides with the DAB solution for 10-15 minutes in the dark and you can inactivate the DAB with water.
We use the DAB kit from Vector.
A light green counterstain could be nice when you work with DAB.
-aspergillie-
QUOTE (aspergillie @ Dec 14 2007, 02:12 AM)
QUOTE (cancerous @ Dec 13 2007, 09:34 PM)
Hi all,
I plan to do some immunocytochemistry with cultured cells on the coverslip. I need to use HRP in the secondary antibody-DAB reaction instead of fluorescent dye-labeled secondary antibody. Can anyone post or upload good protocol for this purpose?
Thank you all in advance.
I plan to do some immunocytochemistry with cultured cells on the coverslip. I need to use HRP in the secondary antibody-DAB reaction instead of fluorescent dye-labeled secondary antibody. Can anyone post or upload good protocol for this purpose?
Thank you all in advance.
After the incubation with yoyr secondary antibody, you have to wash it 3 times (TBS or PBS). Then you make a solution of 5ml TBS (or PBS), add 4 drops DAB and 2 drops H2o2, you incubate the slides with the DAB solution for 10-15 minutes in the dark and you can inactivate the DAB with water.
We use the DAB kit from Vector.
A light green counterstain could be nice when you work with DAB.
Thank you for your answer. I have something to ask you again. After DAB-HRP rxn and conterstaining, do I also have to dehydrate with ethanol (30-50-80-95-100%) and finally with xylene? If so, can you explain that in detail?
-cancerous-