In my experience trying to block deposited endogenous mouse IgG in mouse kidney from being detected with secondary anti-mouse Abs MOM kit did not work. I tried increasing concentration of blocking reagent up to 6 times what they recommended together with increased blocking times, without result. Primary Ab incubation was not important because the Ab was quite good and it worked in 1 h RT or O/N 4 ºC. Of course your level of background will depend on the amount of endogenous IgG in your mouse samples. I have now changed to rabbit mAbs with Jackson´s secondary Abs pre-adsorbed with mouse Ags.
   
  Best regards,
   
  Guillermo

sohail ejaz  escribió:
  I am trying whole mount staining of mouse Retina, Trachea and 20 micrometer tissues with vWF (Rabbit anti human von Willibrand factor polyclonal antibody, Chemicon international) SMA (Monoclonal anti-actin, alpha smooth muscle antibody produced in mouse) and i need to use MOM kit (Vector) for this purpose.

They recommend to incubate the samples for just 30 min with primary antibody.
I wonder if this time is enough..................?? 
Can anyone guide me a good protocol regarding this issue.

Thanks

Dr. Sohail


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