I need some advice on the following experiment. We have fixed cells on coverslips that we need to examine with three specific antibodies. One is rabbit, two are mouse.
I can stain the rabbit with anti-rabbit alexa 488, and I can stain the first mouse antibody with anti-mouse alexa 594. After washing, I also want to have a third mouse antibody, but this antibody (to gamma-tubulin) will be conjugated directly to Alexa 647.
Can I do this? Obviously I am worried about the anti-mouse 594 going to the tubulin antibody, or that some of the tubulin might bind to the mouse 594 at that location. I haven't done 3 (4 with dapi) colour microscopy before....
Dear Michael,
Yes, it is possible to do stainings with antibodies of same species. There are 2 possible ways
1. If you plan to add the second mouse antibody after incubating with the first one, make sure you do extensive washes to remove unbound antibodies. You could even consider reducing the concentration of the secondary antibody (in this case secondary 594)
2. Another option is to check for the isotype of the mouse primary antibodies used. There are isotype specific secondary antibodies. If the primary isotypes are different, you can incubate both of them simultaneously and use specific secondaries.
Hope this helps you.
Meera
Dear Michael,
I usually have good results with protocols and products from Jackson ImmunoResearch (note: no finanacial interest ). They have a section on their website that deals with detecting targets using two antibodies produced in the same species:
https://www.jacksonimmuno.com/technical/products/protocols/double-labeling-same-species-primary
I hope it helps.
Good Luck!
Jill
- Badges
- Science method
- Similar topics
- Biological Science
- Histology
- Staining