[Histonet] BACKGROUND TROUBLE - Rat IgG2b Isotype control
emerald_lake77 <@t> yahoo.com
Thu Dec 14 15:04:33 CST 2006
BACKGROUND TROUBLE USING Rat IgG2b Isotype control
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 Serotecs 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 Vectors 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 doesnt 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.
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