I bought a secondary antibody to use for immunohistochemistry, but the manufacturer says the antibody is only validated for Western Blotting, not Immuno. Because of this, I don't think which dilution to use, and I don't even know if it will work. Has anyone had this problem?
Hi Caroline,
for a secondary antibody there is not that much difference between WB and IHC, in both cases it only has to detect the primary antibody in the presence of blocking buffer.
The main differences are in the Antibody modifications/tags that are used for detection.
I would recommend checking which blocking-buffer was used for WB in the documentation and if it is compatible with the IHC procedure you have, if not stick to the blocking buffer you normally use. Also take along a negative control without primary antibody to check if the secondary antibody doesn't bind a-specifically to the tissue.
Best,
Rob
Hi Caroline
for a WB you split proteins by electrophoresis into different weights. Bands with the wrong weight are most likely false positive interactions of the AB. This is what you cannot distinguish in your cells. I recommend to look at the WB shown in the Antibody documentation and see if there are (many) false positive bands detected.
If that is the case the AB will also detect those in your fixed cells and the signal will be not explicitly derived by your protein of interest.
Best
Hi Caroline,
for a secondary antibody there is not that much difference between WB and IHC, in both cases it only has to detect the primary antibody in the presence of blocking buffer.
The main differences are in the Antibody modifications/tags that are used for detection.
I would recommend checking which blocking-buffer was used for WB in the documentation and if it is compatible with the IHC procedure you have, if not stick to the blocking buffer you normally use. Also take along a negative control without primary antibody to check if the secondary antibody doesn't bind a-specifically to the tissue.
Best,
Rob
I fully agree with Rob Mesman . The jobb the 2ndary has to do is detecting epitopes on the primary antibody bound to the antigen. The protein structure of this antibody is unchanged since most treatments are done before the primary is added, so only affecting your antigen of interest. Normally you use 1-10µg/mL, higher concentrations then in WB, and other labels as well.
However, secondaries for IHC are often preabsorbed leading to no/lower reactions with endogenous proteins. Therefor, have a control section where your primary is replaced by buffer only, but following all other steps, so you will see unspecific binding. This can be reduced by addition of proteins (IgG-free BSA) or serum (in some cases I add 30% serum). Still some secondaries bind to much in IHC, and then I just buy/use another, which is cheaper then figuring out why.
Good luck!
Olav
Hi, people. Firstly, thank you for the answers.
I found an article that used the same secondary antibody, for IHC as well, at a 1: 1000 dilution. I just did it at this dilution too and there was a float mark on my tissue (rat brain). Now, I will make a more concentrated dilution (1: 200) to see if the result will be better. My secondary antibody is polyclonal and specific to my primary antibody (purchased from same company, inclusive). Do you think I should still do a negative control without the primary antibody? Thanks.
Yes, always! But since you're working on rat, you do it once, for every kind of tissue you use, for every secondary you use. My experience from different mice strain is that there are binding differences there as well. Normally I work with human material, and then I test every biopsy for non specific binding of 2nd and 3rd layer I use. When I shift using another 2ndary, I test have to that again on that block. Additionally I might use IgG-control sera (e.g. purified mouse IgG2b) for testing the non specific binding of the nature of my primary antibody. They sometimes give huge variation in "background", and are not purified in the same way as the primaries, so in IHC I do not think they are the best controls. Rather use a primary with similar isotype and purification, but different staining pattern, or not expressed in that tissue. Sometimes you have internal controls already, in another primary used on the same tissue. BTW: most secondaries are polyclonal; they are cheaper to produce (bigger animals) and can find several epitopes on your primary, increasing signal. If they are of "whole IgG" type, the can have physically more space for more labeling as well.
Good luck!
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