Hello Hello,
I am trying to stain DsRed/GFP lymphocytes in recipient tissue. Tissues are frozen spleen embedded in OCT gel.
I used a primary Rabbit polyclonal to RFP (Anti-RFP antibody ab62341) against RFP to stain the cells.
The problem is it doesn't work.
Tissues were fixed with either acetone or PF 4% and then permeabilised with 0,2% triton.
Pre-blocked with normal mouse serum.
Primary AB concentration 1:100 or 1:200.
Sec AB: Alexa fluor® 568 goat polyclonal to rabbit Ig
Dear Sina.
There could be many reasons, why you see no staining:
- No lymphocytes in recipient tissue present
- No Ds-Red in lymphocytes present
- Wrong filter sets in your microscope (should be excitation max 578, emission max 603 nm)
- Staining protocol issues
Regarding the last point, you could try this:
- Block with normal goat serum instead of mouse serum. If you try to stain mouse tissue and block with mouse serum, then probably you block everything. Use serum from the host animal your secondary AB is made in (in your case: goat).
Also there are informations missing, which could be helpful for solving your problem. Maybe you can share with us f.e.:
- Is your tissue fresh frozen or fixed?
- Is it mouse tissue?
- How long are your incubation times for postfixing (if you use fixed tissue, then maybe you don´t have to fix it again - too much fixation could mask your antigen), primary and secondary?
You could also try the link below (abcam´s site offers staining protocols and references):
https://www.abcam.com/rfp-antibody-ab62341.html
Hope this is helpful.
Dear Sina.
There could be many reasons, why you see no staining:
- No lymphocytes in recipient tissue present
- No Ds-Red in lymphocytes present
- Wrong filter sets in your microscope (should be excitation max 578, emission max 603 nm)
- Staining protocol issues
Regarding the last point, you could try this:
- Block with normal goat serum instead of mouse serum. If you try to stain mouse tissue and block with mouse serum, then probably you block everything. Use serum from the host animal your secondary AB is made in (in your case: goat).
Also there are informations missing, which could be helpful for solving your problem. Maybe you can share with us f.e.:
- Is your tissue fresh frozen or fixed?
- Is it mouse tissue?
- How long are your incubation times for postfixing (if you use fixed tissue, then maybe you don´t have to fix it again - too much fixation could mask your antigen), primary and secondary?
You could also try the link below (abcam´s site offers staining protocols and references):
https://www.abcam.com/rfp-antibody-ab62341.html
Hope this is helpful.
Please use a positive control such as a transfected cell line to determine if there is any problem with your antibody.
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