I am trying to do laser capture for immune cells that infiltrate a subcutaneous tumor that is grown in a Matrigel plug in a mouse. However, my antibody seems to be sticking nonspecifically to a lot of tumor cells, and I can't get nice staining of my CD3s. I think this is because of the Matrigel because my anti CD3 antibody is my workhorse that always looks nice in every other scenario I've tried it with. Since I want to get decent quality nucleic acid out of these CD3s, I feel like my options are limited for what I can do to block the nonspecific binding. I don't want to introduce a lot of RNAses and DNAses with blocking reagents like serum, and I don't want to degrade the nucleic acid with excessive heat.
The other problem is that this particular tumor type I am interested in does not form nice solid tumors subcutaneously without matrigel, so I don't think I can just axe that from the experiment either.
Any suggestions?
Hi Virginia,
do you think the antibody is binding to some matrigel-components? In this case you could try to do a pre-adsorption of your antibodies with matrigel-solution prior to incubate your tissue-sections. That should help to increase the specificity!
Hope that suggestion is helpful,
best regards,
Christian
Hi Virginia, just to add to Cristian's suggestion, you could try adding a mixture of excess unlabeled antibody that has the same isotype as your CD3 antibody.
Also, you could try double staining using anti CD3 and anti CD45 to identify leukocytes.
You can exclude epithelial cell by using anti EpCAM and anti cytokeratin.
You can exclude endothelial cells with anti CD31 and anti CD146.
You report staining of the tumor cells, but not of the matrigel, which seems a bit odd for the reaction being due to the matrigel. Have you tried just diluting down your primary ? Also, there are blocking solutions available that are serum-free (eg. from Dako).
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