I'm trying to stain mouse blastocysts with antibodies for Cdx2 and Oct4, I've had great success with my Cdx2 antibody however the Oct4 staining is always hazy and non-specific. I'm using an antibody that is published in many mouse embryology papers: Santa Cruz Oct3/4 (C-10). I've tried different dilutions of the primary antibody and I've tried an IgG specific secondary as well as a polyclonal secondary with the same poor results. I don't think it's my protocol since I've used it successfully with other antibodies. Is it possible I just got a bad antibody batch since others have used this antibody successfully? I'd appreciate any recommendations, thanks!
Dear Laramie,
Kindly check out this protocol,
Indirect immunofluorescence
Embryos were freed of zona pellucida using acid Tyrod’s solution
(pH2.5, [11], fixed in 4% PFA in PBS (without Ca2+ and Mg2+ )
for 30 min at room temperature, and then permeabilized with
0.3% Triton X-100 in PBS for 20 min. Blocking of nonspecific
binding of antibodies was performed by incubation in PBS/ 3%
BSA/0.05% Tween, overnight, at 4C. Embryos were then
incubated overnight, at 4C, with the primary antibody: antiOct3/4
(mouse monoclonal IgG, Santa Cruz Biotechnology, USA,
0.75 mg/ml) at 1:100 dilution or anti-Cdx2 (mouse monoclonal
IgG, BioGenex, USA, 0.75 mg/ml) at 1:50 in PBS containing 3%
BSA. After being washed (3×15 min in PBS) embryos were
incubated with the secondary TRITC-conjugated anti-mouse
antibody (Jackson ImmunoResearch, 0.75 mg/ml) at 1:200
dilution in PBS/ 3% BSA, for 1 h, at room temperature and then
washed once in PBS. Negative control was prepared by omitting
the incubation with the primary antibody. Chromatin was stained
by incubation of blastocysts in DRAQ (10 M; Biostatus,
Leicestershire, UK) for 10 min. Embryos were imaged with
Zeiss510 confocal laser microscope.
Reference : http://repbiol.pan.olsztyn.pl/docs/pdfs/repbiol_vol11_num2_page145.pdf
Regards,
Sanketh Dhumal S
Is it possible I just got a bad antibody batch since others have used this antibody successfully?
Maybe yes.
May be the batch is bad or the timing!
https://www.ncbi.nlm.nih.gov/pubmed/21804635
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