I am running a fluorescence IHC to detect calreticulin in paraffin embedded tissue.
Primary antibody is polyclonal calreticulin and secondary antibody is Alexa Flour.
Using 0.05% Tween 20, 3% goat serum and 3% BSA as a blocking buffer.
can I use this buffer to dilute the primary and secondary antibody?
Thank you
Technically there is nothing wrong with using the buffer you are listing above for both the primary and secondary antibody ( I am assuming this is in PBS or TBS). However, if you bought the antibody, please check the supplier information as well. They might specify a buffer system for use in IHC.
I have the same question and I am thinking that maybe Diluting in buffer that has BSA is not so much a good idea because my theory is that BSA might bind the primary ( scavenge it) NOTE: This theory is not based on evidence, just a thought.
Having that said and assuming it is possible then maybe it is a good idea to never dilute in BSA rather only use BSA once and as pre blocking step then wash the cells with non BSA buffer before incubating with the primary.
I need your opinions please on this idea?
Much appreciated
Thanks.
Regarding Mohammed Pharaon question. I think that using buffers with BSA to dilute antibody is not a good idea, since secondary antibodies may bind to BSA or serum therefore reducing antibody affinity.
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