immunohistochemistry - (Feb/13/2020 )
Hello,
I am a PhD student. I am planning a small study that will use the immunohistochemistry technique. But I don't know how to choose the primary antibody to use.
I took paraffin sections using rat liver samples. I search the internet for companies primer antibody that I will use. For example; abcam, santa cruz...
What should I look out for in polyclonal, monoclonal, conjugated, unconjugated, host species,or description sections?
I have anti rabbit as secondary antibody. Do I have any importance for this primary antibody?
In short, what are the things to consider when choosing antibodies for immunohistochemistry?
Since your secondary is anti-rabbit, your primary must be from rabbit. And since monoclonal are mostly prepared in mice, your primary will most likely be polyclonal.
your primary should be against your protein of interest, if available. It won’t need to be conjugated with anything (that’s usually for secondary antibodies unless you’re not using a secondary antibody).
you should also look at the data sheet to see for which techniques the antibody is suitable. This is not absolute but will give you an idea if you should be successful.
thank you very much
How much important is the brand of the antibody? I found it in Biorbyt, Thermo Fisher Scientific, OriGene Technologies and Bioss brand.
I found abcam antibody. but the application writes ELISA and WB. Datasheet can be applied for immunohistochemistry, but there is additional information that the user determines the dilution. Can I run Abcam brand antibody?
In general, the brand is unimportant (although there is one brand that many find unacceptable for many of their antibodies, i can’t remember which one). The dilution required for use, as stated in the data sheet, is a guideline, the optimum dilution should always be determined by the user. We found that most of the time (with our homemade antibodies) immunohistochemistry required about 10X the concentration required for western blot.
mdfenko on Sat Feb 15 20:05:30 2020 said:
In general, the brand is unimportant (although there is one brand that many find unacceptable for many of their antibodies, i can’t remember which one). The dilution required for use, as stated in the data sheet, is a guideline, the optimum dilution should always be determined by the user. We found that most of the time (with our homemade antibodies) immunohistochemistry required about 10X the concentration required for western blot.
Thank you very much.
I have rabbit seconder antibody. I understood that I should look for a rabbit in the primary antibody. But now I found Recombinant Rabbit Monoclonal Antibody. What does mean recombinant monoclonal? I ask this because it is usually only written as monoclonal antibody. Which antibody should I choose in this case? (polyclonal or monoclonal)
Recombinant is made by transferring the genes for antibody production into another organism which may be easier to grow than the original cloned animal cell (this is a slight oversimplification).
the concern with monoclonal antibodies is that it is specific for an epitope that you know to be present and accessible in your situation. Polyclonals will be to multiple epitopes and will have a better chance of binding to the protein of interest at more than one site. monoclonals will be more sensitive to a specific site.
In addition to mdfenko's comments. You should use a polyclonal - monocolonal antibodies are usually generated against a specific epitope on the protein using a short peptide (usually 8-20 amino acids long), this may or may not be on the surface of the protein when it is all folded up, like you will have in your IHC. Polyclonals are usually generated against the whole protein and contain several epitopes withing the antibody population.
As you have paraffin sections, most likely you will be doing some sort of antigen retrieval step during your IHC - this is basically a protease digestion or something similar that degrades some of the surface proteins which are all bound up with the fixative, so that the antibody can access them. If it so happens that a monoclonal antibody targets a region that is particularly susceptible to degradation by antigen retrieval, then you won't see anything. A polyclonal gets around this difficulty by having multiple epitopes.
Long answer short: use a polyclonal.
Oh - the company mdfenko mentioned to avoid is Santa Cruz Biotechnologies. They have a history of selling antibodies that have not been tested at all. On the other hand I have had great success with one of their mouse anti-actin monoclonals for western blots at concentrations less than 1:50000.
This is the first time I am planning a study on my own. so ı am so excited.
Choosing antibodies was really scary for me.
thank you very much for your answers.