Antibody-Based Technologies => Antibodies Review => Antibody P => Topic started by: marina77 on January 15, 2008, 12:29:35 PM

Title: PCNA
Post by: marina77 on January 15, 2008, 12:29:35 PM
dear all,
i have a  policlonal antibody rabbit anti-rabbit PCNA (Lifespan Biosciences). The reactivity is dedicated in rabbits. I can hardly beleive in good results:((( Will i have to high bacjground?
Tnaks
Title: Re: PCNA
Post by: richard03 on January 15, 2008, 07:29:15 PM
Please post more details about your protocols so we can help.
Title: Re: PCNA
Post by: marina77 on January 16, 2008, 05:58:00 AM
Matherial: rabbit cornea
Detection of proliferating cells on paraffin-embedded slices

1. Deparaffinization
2. Descending concentrations of ethanol and xylene , Aqua dest
3. Rinse 3x 10 min in PBS, pH=7,4
4. Antigen retrieval (boiling in cytrate buffer, pH=6 for 5 min)
5. Rinse 3x 10 min in PBS, pH=7,4
6. Blocking for 1 hour (PBS, 2%NGS, 1%BSA, 0,3% Triton-X-100)
7. Primary antibody dilutes in PBS+1%BSA+0,1% Triton incubation ON (rabbit anti-rabbit PCNA, Lifespan Biosciences, concentration is npt determined yet)
8.  Rinse 3x 10 min in PBS, pH=7,4
9. Secondary antibody, goat anti-rabbit-Cy3 (1:500, Chemicon)
10. Counterstaining with DAPI


Title: Re: PCNA
Post by: richard03 on January 16, 2008, 07:37:40 AM
I have a few comments on your protocol.

1) Antigen retrieval should be longer. Try 20 minutes.
2) Cut primary antibody incubation time to 1 hour at room temp. If background is high, you may reduce concentration.
3) The background may be due to autofluorescence. Check the slides before staining to confirm.
Title: Re: PCNA
Post by: marina77 on January 16, 2008, 02:00:58 PM
Thank you very much for yor comments.
And what about "rabbit anti-rabbit" in rabbit? 
Title: Re: PCNA
Post by: richard03 on January 16, 2008, 06:38:24 PM
Sorry that I missed this important point. Yes. There will be background problem for using rabbit antibody on rabbit tissue.

You can either use a NON-rabbit antibody or use labeled rabbit antibody (i.e. HRP or FITC labeled).

Thank you very much for yor comments.
And what about "rabbit anti-rabbit" in rabbit?