Hi all,
It would be greatly appreciated if someone can please share a protocol for double staining of Intracellular marker (FOXP3)/(extracellular marker) CD3 on paraffin embedded section for Immuno-fluorescent staining.
Can I please ask specifically whether you permealise the tissue and when this step takes place before or after adding the surface marker?
Thanks
Fatma
well, i think it might not be very easy to do CD3 staining on parrafin embeded tiuuse. but you for sure, you should do first CD3 (surface staining) and then permeabolize before FOXP3 staining.
Thanks a lot Muhammed for adding your answer, really appreciated
can I just please clarify, would you do all the steps for surface staining adding primary then secondary (in my case for IF will be flurochrome conjugated) then do the permealisation? right?
No, if i wanted to do, i will do surface staining with primary, the permelize, do intra-cellular staining and at then end... 2ndry for both together...
I agree with Muhammad you should do first surface staining and then permeabolize and intra-cellular staining.
Thanks a lot Morteza for ur input, appreciated
Do u please have a full protocol to share?
The idea of permeabilistion is to allow the antibody to enter into the cell cytoplasm and nucleus. Apparently, in 3-4 um thick tissue section the organelles and the membrane are all already exposed! The important thing with your paraffin section is to dewaxing and antigen retrieval. Do it very well.... keep the sample at least for 15 min 2x in acquaclear then follow the standard rehydration process. Then do antigen retrieval in 10mM citrate buffer in microwave for 12 min. Next crucial thing is to block the sample. I will go with with 1% BSA or 10% FBS and 0.2% triton x-100 in PBS for 1 hour at room temperature incubation. I will chose FOXP3 and CD3 raised in two different species, preferably, CD3 could be conjugate with either FITC or TRITC. I will start with one ab first to optimise the dilution in above buffer in 4 degree overnight incubation followed by secondary (usually 1:200 to 1:500) at RT for 1 hr. Once both antibodies are optimised, I will do primary ab treatment with both ab (CD3-conjugate/FOXP3) together then secondary for FOXP3 with DAPI and then mount with Prolong Gold antifade without DAPI. This approach would be my FIRST GO! If it doesnt work then you have to play with blocking buffer and need to stain the sample separately with two different antibodies. Other thing, please bear in mind that paraffin sample is sometime problematic for fluorescence IHC. Non-specific binding is the main problem to overcome. Hope this help. Good luck.
Hi Khondoker, Many Mannnnny Thanks, I will give it try and let u all know.
Hi,
Thanks all for ur inputs, I had considered them, and here is my optimised protocol
1-Dewax 10min in Xylene twice
2-H2O2 20mins
3-Ag retrieval in Citrate buffer in microwave for 10mins
4- Glycine for 10mins
4-Blocking for 10mins in SFB (serum free blocking) buffer
***5- Incubate Surface marker targeting Abs (CD3) in 5%SFB in PBS overnight at 4C
***6-next day, incubate with intracellualr marker Abs (FOXP3) in 5% SFB and 0.2% triton x-100 in PBS for 2 hour at room temperature.
7- Wash and add secondaries
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