I am looking for a CD-31 protocol:
1- Protocol (Antibodies, concentration, fixation, etc..)
Thanks in advance!
Hi, for mouse tissue I had the best experience with rat anti-CD31 antibody from BD Biosciences ( Clone MEC 13.3), Dilution 1:50. For rat tissue I would rather recommend to use anti-RECA-1 antibodies e.g. ( Mouse anti Rat RECA-1 antibody, clone HIS52).
Watch out to not overfix the tissue, for brain it needs to be around 4-5h of post-fix. Hope this helps!
Cheers,
Ruslan
Hi Ruslan,
Thank you for your help! I really appreciate it!
Regards,
Luís
Hi Tai,
I going to do a test this Monday but I am doing a double stain with Dystrophin. Below is the protocol I am using right now. But for CD31 this protocol works great!
Antibodies:
CD31(R&D Systems AF3628) (7.5 ug/ml) (37.5 ul of CD31 antibody in 1 ml of PBST + 5% NHS = Final Concentration 7.5 ug/ml)
Dystrophin (H5) conjugated with AF488 (Santa Cruz - sc-365954 AF488)
Procedure:
1- Acetone 100 for 5 min at - 20C (Pre-cooled Acetone)
2x5 min in PBST (Wash)
Blockin Solution:
5% NHS at RT for 30 min
3x5 min in PBST
Primary antibodies: (Protect from light)
CD31- (37.5 ul in 1ml of Blocking Solution (PBST + 5% Normal Horse Serum)
Dystrophin (H5) 1:250 in Blocking solution (PBST + 5% Normal Horse Serum)
OBS: If you would like to do a double stain you must mix both antibodies in the same tube!).
Negative control: Only Blocking solution
Incubate overnight at 4C
Next day:
3x5 min in PBST
Secondarie antibody in Blocking Solution (PBST + 5% Normal Horse Serum)
CD31 - 555 IgG (1:500 in Donkey Anti-Goat IgG)
Negative Control: Only the Blocking Solution!
1h of incubation in RT
3x5 min in PBST
Coverslip with Prolong Gold ( NO DAPI!) (Life Technologies P36930)
Let air dry for 24h in the dark
Store at -20C
If you have any questions, please let me know!
Sincerely,
Luis
Hi Luis,
Thanks for the detailed protocol. I think my stains finally came out adequately. How have you been quantifying your CD31 cells. some paper report average CD31 cells per area, while some average cd31 cells per muscle fiber. Sometimes they clump together especially near the injury sites. Just out of curiosity, what is your animal injury model? We use VML model.
Tai
Just an update:
Instead using 1h, I am using 30 min on secondary!
I count the total number of capillaries and divided by the total number of fibers. Total Capillary /Total Fibers. I am using the Image J to count the number of Capillary and fibers. Another way to count the fibers is using either Myovision (University of Kentucky) or Myosoft plugin for Image J (Encarnacion-Rivera, L - 2020). You can use area as well it depends of your question. Either way works!
We are using Fisher Norway Brown 344 (Rat) and C57BL6 (mouse).
Let me know if you have any other questions!
Best,
Luis