Dear all,
I am in search for ideas for what might cause the following issue:
I have done IHC-IF extensively and wanted to perform stainings on cultured cells now, too. However, I am observing unspecific staining of all primary antibodies tested. It is a mystery to me!
Here are the details:
I am staining murine cell lines that have been fixed with 4% PFA for 10 Minutes.
Protocol (washes performed, but omitted here):
- No retrieval
- Blocking for 60 Minutes in PBS with 5% NDS + 0,3%Tx-100
- Primary AB in PBS + 3% NDS+0,3% Tx-100 over night at 4°C
- Detection with Donkey anti Primary 1:500 in PBS + 3% NDS (60 Minutes incubation at RT)
Results: Three different cell lines (neuronal, astrocytic, endothelial) all stain equally for the antibodies used (broad variety from different species), e.g. anti-GFAP, anti-GFP, anti-MAP2... Most antibodies tested work well in IHC (some are untested) and - based on the literature - many of them are used in ICC successfully. A control without primary antibody does not show staining, so autofluorescence or unspecific binding of the secondary antibody do not seem to be the issue. The composition of the media the cells were cultured in differ between the lines.
Steps taken so far:
- Used new buffers, aliquots, cells etc.
- I have done a dilution series with two different antibodies:
- Antibody A should only stain cell Line A and antibody B should only stain cell line B.
- Both cell lines were stained with both antibodies in 11 dilutions ranging from 1:500 to 1:512K
- Result: Both stainings A and B do not differ between cell line A and B. One can see that the intensity is clearly reduced with lower concentrations of the primary antibody, but there is signal. This signal is absent in the control without primary antibody.
I have also planned to do a qPCR next week to verify the identity of the cell lines just to be sure, but I am feeling like it is a technical problem. I cannot come up with a reason that makes the specimen "sticky" for different primary antibodies, but not for secondaries. It seems unlikely to me that there is a problem with all primary antibodies tested, especially since they work well in free-floating slices.
Any ideas what might cause it and how to solve it would be very much appreciated!
Henrike
Hi Henrike,
You blocked the antigens with donkey serum so in the control group, the secondary antibody won't have non-specific bindings. However, this did not rule out the possibility that the primary antibodies have non-specific bindings. Is it possible to use siRNA or certain approaches to deplete the target protein as a control?
Hope this helps.
Yuning
Dear Yuning,
thank you for your suggestion! Unfortunately, the problem exists across all antibodies and cell lines tested. Some of these certainly should not react, e.g. anti-GFP in cell lines that do not express GFP. One could consider this a control similar to your suggestion - indicating that there is a more general issue which I cannot pinpoint.
Best regards
Henrike
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