I am TAing a section of a class for Toxicology masters students to learn about immunofluorescence. The class is split into two sessions one week apart. The idea is to fix the cells, block, and add the primary antibodies one week, then add the secondary antibodies and look at the slides the second week. Is one week at 4 degrees too long to incubate with primary antibodies? Would it be better for me to remove the primary solution after the students leave, leave the cells in PBS, and replace the primary antibody solution the day before they come to do the secondary?
Hello
Kindly look at the links.
Good Luck
http://www.abcam.com/protocols/immunocytochemistry-immunofluorescence-protocol
http://www.sciencegateway.org/protocols/pdf/ihc_icc_protocol_guide.pdf
That is a good question. I think the longest I ever had the primary antibody on sections, has been over the weekend at room temperature. It still worked. However, what does not seem to work is storing diluted antibodies at 4C and using them after a week. Antibodies can degrade (Anal Chem. 2013 Jan 15;85(2):715-36.; PLoS One. 2014 Jun 24;9(6):e100736.) but whether chemical reactions involving certain amino acids is the driver of the the degradation is not clear to me.
So, would I do incubate the primary antibody for a week? No. Is it possible? Maybe. Maybe it depends on the antibody itself and whether important amino acids are prone to the degradation process as described in PLoS ONE. 2014;9:e100736.
If you plan such an experiment, I would strongly recommend that you test this beforehand. It is not the standard procedure.
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