Thank to anyone spear their attetnion to my question.
I am working on flurence fluorescent immunostaning staining CSF1R in both paraffin-fixed and PFA-fixed human glioma samples. I am faced with problems that current avaibale commerical antibodies are not working at all, no matter using TSA-enhancement or bearing antigen retrieval steps.
Please help if your are experineced with CSF1R staining protocol in human tissues. Any tips on experimental steps or recommanded antibodies will be greatly appreciated, thank you.
Hallo, there are two reasons why your antibody is not working. Either it is not suitable for human tissue and/or not for that kind of application you like to use it.
In both cases it is helpful to check the datasheet of these antibodies.
I have no expirience with CSF1R-antibodies, but I found one from the company abcam (cat#ab 313648). Maybe it could work. Good luck!
Hello, Zijian Zhou .
Unfortunately, MedChemExpress is currently unable to provide CSF1R antibodies for IF-tissue/IF-P. However, Our laboratory has verified that CSF-1R Antibody (YA1299) and CSF1R Antibody (YA3685) are suitable for paraffin section IHC-P experiments (see the attached figure for details).
Our technical colleagues will give you some suggestions on operation, hoping to help you.
1. Antigen retrieval is a key step for the success of CSF1R immunofluorescence staining. The antigen retrieval buffer we recommend is as follows:
Tris-EDTA buffer (pH 9.0): suitable for heat-mediated antigens, which require 20 minutes of retrieval.
Citrate buffer (pH 6.0): alternative, suitable for some antibodies.
TE buffer (pH 9.0): can also be used as an effective antigen retrieval buffer.
2. If the signal is still weak after optimizing the antigen retrieval reagent.
It is recommended that you directly increase the concentration of the primary antibody or extend the incubation time. Or find the cause from the sample, and check whether the tissue fixation time is too long to cause antigen burial.
3. Find the cause according to the elimination method and prescribe the right medicine.
Use known CSF1R-positive tissue as a positive control, such as normal spleen or lymph node tissue, to test whether it is a problem with the antibody product.
We also collected a published article (Claudia Foray, 2022). The author fixed the mouse brain tissue with 4% PFA overnight, embedded it in paraffin, and made coronal sections. And double-labeled with CSF1R/Iba1 and TSPO/Iba1 fluorescent antibodies. Detailed experimental operations can be viewed with the following literature link:
https://jnm.snmjournals.org/content/63/9/1386.abstract
If you have other experimental operation details, you can also post them for discussion. For example, your fixation conditions and tissue processing steps.
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