I would like to label p21 positive cells in irradiated mouse embryonic cortex. I am currently using an antibody from Cell signaling that is directly coupled with the Alexa Fluor 488 (Ref 5487). Although it never seems to work, either in FACS or on laminine-coated mouse cortical neural stem cell culture, nor on mouse brain paraffin sections. It seems that most of the antibodies against p21 work in all cell types, except in mouse cortical neural stem cells. Indeed I have tried it on human cortical neural stem cells where it seems to work.
Do you have any experience with p21 staining in mouse cortical neural stem cells? Do you have any advice regarding tissue fixation, reagents for immunostaining or specific antibody against p21? I plan to idenify p21 positive cells using FACS and laminine-coated mouse cortical neural stem cell culture, and brain paraffin sections.
Amandine, You seem to be using this antibody in a number of experimental contexts and each requires a different answer:
1) What is the outcome you require from FACS? p21 is an intracellular marker, so the antibody cannot be used for obtaining live cells since fixation is required for intracellular markers for FACS. Additionally, only very few people have reported extracting RNA of quality suitable for qRT-PCR or microarray from FACS sorted fixed cells. It would work in characterisation of changes in cell populations using FACS. You need to determine that the antibody is suitable for FACS on the antibody datasheet. The Biocompare comparison website is a good way to find a p21 antibody that is suitable for FACS. Look for one where the fixation protocol is stated.
2) Immunocytochemistry of cells grown of coverslips.Find a suitable antibody for immunocytochemistry/immunofluorescence on Biocompare - it may tell you which fixation technique to use (4% paraformaldehyde/PBS at room temperature or methanol/acetone at -20ºC. Fixation and antibody dilution information may be on the data sheet or in the papers cited on the datasheet.
3) Much of what is siad about immunocytochemistry applies to paraffin embedded sections. You also need to know whether the sections need an antigen retrieval technique (e.g. boiling citrate buffer pH 6.0 for 15 min) for the antibody to detect the antigen.
In general, you need to buy antibodies that are known to work for the particular staining method you require. The Biocompare website is a great help for finding antibodies that will work for your particular experiment.
Dear Kenneth,
I really thank you for your detailed answer.
1/ In the FACS analysis the cells have been previously fixed and then stained with antibodies against EdU and BrdU. For those two thymidine, I have no problem of detection.
2/In cells that have been cultured on laminine I have checked the fixation on the antibody datasheet and tried different type of fixation (exactly those that you mentionned).
3/One of my colleague is used to do staining on paraffin sections. After checking the datasheet of the antibody I has made the immunostaining by following the protocol suggested by the manufacturer.
Consequently, it seems that I have already checked all the points that have have suggested. I have already visited a website where antibodies efficient is discussed and from which I have choosen the antibody from Cell signaling.
From here if you have any more advices, it will be great.
Yours sincerely
Amandine
1) Be careful with BrdU staining as harsh denaturing techniques (e.g. HCl) are often used to expose the BrdU nucleic acid antigen A protocol including Dnase may be better than the hydrochloric acid approach.
2) I also suggest a dilution series of the primary when optimising the staining.
The antibody you are using is against HUMAN/MONKEY p21.
Try sth against mouse p21.
Regards!
It is a mouse anti p21.
I have recently solved the problem. Now I used a more diluted concentration of the antibody.
Thanks for the help.
Amandine
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