[Histonet] BACKGROUND TROUBLE - Rat IgG2b Isotype control

[GT Hebert]

BACKGROUND TROUBLE USING Rat IgG2b Isotype control
   
  Hello,
   
  I am staining mouse aortic sinus sections (fixed frozen sections, 10microns) and just received a huge amount of background on my negative control sections.
   
  The antibodies I am using are Serotec’s MOMA-2 Ab and the corresponding isotype negative control, Rat IgG2b.
   
  The protocol I am using is as follows:
   
  Remove OCT using PBS (2 changes, 6 mins each)
   
  0.3% hydrogen peroxide in water to block endogenous peroxidase (1/2 hour room temp)
   
  Either serum (according to Vector’s ABC Elite kit – Normal Rabbit) or non serum block (DAKO) for 20 mins at rm temp.
   
  I tap off both blocks and dabbed with gauze any excess liquid.
   
  Then I add primary antibody (5ug/ml) at room temp for two hours.
   
  I wash three times in PBS + tween (0.05%) – 3x – 5 mins each.
   
  After wash, I continue following protocol for Vector ABC elite kit.
  -         Add secondary Rabbit anti-rat BIOTIN (1/2 hr, room temp)
  -         Wash as above (PBSt)
  -         Add ABC reagent (1/2 hr room temp)
  -         Wash again as above (PBSt)
  -         Apply DAB chromagen (DAKO)
   
  Primary antibody work great – lesions in aortic sinus showed nice staining (macrophages/foam cells) and there was little background throughout the entire sample.
   
  However, my negative turns COMPLETELY BROWN.  I ran the negative at 5, 10 and 20 ug/ml – there is definitely a difference between lower and higher concentrations.  I stopped the chromagen after 5 minutes.
   
  This doesn’t make sense to me.
   
  Can anyone explain what might be happening and the best way I may be able to resolve this problem, making my negative become a negative again???
   
  Thank you all in advance for your helpful advice.
   
  Sincerely,
   
  Gustave H.
  Scientist II
  Wyeth Research
Cambridge MA
 
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