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Optimising

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Alice:
Hello,

I am pretty new to IHC and I have one or two questions.
1. When optimising do I use the positive control tissue to optimise against or the experimental tissue?
2. There are different techniques suggested as to how to dilute the primary antibody - do I use PBS or the blocking serum? I am using the ABC elite kit and there dosen't appear to be a lot of blocking serum made available.
3. Is anyone familiar with COX 1 antibodies from Santa Cruz (anti goat) I am using this on mouse small intestine - when using the blocking peptide there is still staining - any suggestions?

Thanks

JLI:
Hello.

I have a few comments you might use.

1) A very good question. Itīs a good idea that your positive control contains some tissue that will stain like your experimental tissue or the structure you are looking for in your experimental tissue. For instance it is known that some tumors will have fewer epitopes than their "normal" counterparts. This means that you could have negative results on your staining of tumor despite a perfect staining of your normal tissue used as positive control.

3) Have you remembered to block endogenous biotin? Another option is to use a biotin free detection system.

Jan

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