Please help to solve the problem with immunostaining of melanoma samples
A group of us here have been trying to stain melanocytes with an IgM
antibody that we generated. So far, we have observed beautiful staining of
melanocytes in normal or benign (i.e. nevi) skin samples with a fairly
simple protocol -- dewaxing, dehydration, protein block (5-15 min), primary
(1-3 hrs at RT), secondary-HRP (30 min at RT) and detection (note: no
antigen retrieval). However, whenever we tried the same protocol on
melanoma samples, we noticed considerably higher background almost
everywhere. Lower incubation temperature or lower antibody concentration
didn't help much, either
Higher background is probably a consequence of non-specific staining of the
inflammatory or necrotic tissues in melanoma samples by some impurities in
the ascite fluid that we are using. Therefore, it appears that three
factors could be influencing our staining unfavorably:
1) Malignant skin tissue that tend to have higher background;
2) Nonpurified ascite fluid that might contain many other impurities;
3) An antibody of IgM isotype that is often more troublesome than the more
Currently, we are trying to purify the IgM antibody from ascite fluid,
which is a bit tougher than we anticipated. As an alternative, we are also
growing the hybridoma hoping to get some supe for testing. However, we
might also run into the same problem with the supe if IgM or melanoma
sample is the problem.
This leads to my questions:
Is IgM really a problem for immunohistochemical staining on tissue
If so, what can we do except to generate another hybridoma line?
If no, how can we remove the non-specific staining besides incubation
of the tissue section with protein block prior to the primary antibody
(that we have tried)?
Any suggestions or comments will be greatly appreciated.
By the way, I just recently signed on to this newsgroup. If you have
answered these questions in previous posts, I would certainly appreciate it
if you could please forward a copy of your response to me.
Thank you very much for your help in advance, and have a great day!
Yun-Fu Hu, D.V.M., Ph.D.
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