Dear Teisha,Because you gave us only a very few details I have to speculate a bit about the cause of your failing second primary. Paul's suggestion about the second staining sequence is fully correct. I would like to add that I believe this only happens when the two epitopes of interest are really very close together. The only time we saw this actually happen was when we tried to combine CD3 and the gamma-delta Tcell receptor, at the time that it was unknown that both molecules belongs to the same complex. Perhaps more likely is that you are performing a so called 'sequential' type of double staining that starts with a complete staining procedure including HRP activity development with DAB. Because the DAB reaction product shields so effectively one can perform a second staining sequence (under ideal conditions) without any cross-reaction problems. Even with two mouse primaries. As far as I know DAB is the only enzymatic reaction product that has this 'sheltering' characteristic. Sometimes this sheltering effect also blocks the second antigenic site. Mostly when the two epitopes are very close together, but sometimes also just at random. In either case you end up with unwanted single-staining as you described. As a solution to your problem, you can try to stain the first epitope with a red alk. phos. reaction product, then perform a second HIER step for only 10 min at 98C (in a buffer that fits best with your second epitope) and perform a second staining sequence ending with a blue alk. phosp. reaction product, or whatever chromogen you like. The second HIER step removes and/or destroys your first primary antibody and detection reagents, but leaves the red alk. phosp. reaction product unchanged.Hope this helps a bitChris Chris van der Loos, PhD
Dept. of Pathology
Academic Medical Center M2-230
Meibergdreef 9
NL-1105 AZ Amsterdam
The Netherlands
Date: Mon, 14 Jul 2008 12:59:21 -0700 (PDT)
From: Teisha Robertson
Subject: [Histonet] double staining
To: h n
Good Afternoon,
I am double lableling using mouse antibodies. My single staining works fine but when I double stain with the second primary anitbody I receive no response. I am using G-protiens if you were wondering. Can anyone suggest a protocol? Any suggestions given would be greatly appreciated.
Teisha
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