[Histonet] Isotype background

[Merced M Leiker]

I wouldn't get rid of the isotype control altogether; it's fulfilling it's 
purpose in trying to tell you something. Have you tried a no-primary 
control in parallel with your isotype control to see what the secondary is 
binding to - the tissue or bound goat IgG? What type of tissue is it? Maybe 
the goat IgG is binding to Fc receptors in the tissue. Try an Fc block. Or 
try a different goat IgG or one from a different company.

But it may not even be a big issue; your goat IgG is not giving you the 
specific stain pattern that you are observing with your primary, right? 
It's just giving a lot of background? Instead of spending a lot of time 
troubleshooting the high background issue with your isotype control, it's 
already succeeded in telling you that your primary is specific, which is 
what you ultimately wanted to know, so you could just take that for what 
it's worth and go from there...

Just my two cents' worth!

Regards,
Merced

--On Tuesday, October 13, 2009 4:48 PM -0500 "Adam ." <anonwums1 <@t> gmail.com> 
wrote:

> Hi all,
>
> I am trying some IHC, and I am having a peculiar problem. Like I expect,
> my antibody of interest (anti-mouse goat polyclonal) stains
> nonspecifically at high concentrations (10 ug / ml) but as I titer it
> down (3 ug / mL), it seems to stain relatively specifically the cells I
> think it should stain. However, at 3 ug / mL, my isotype goat IgG stains
> nearly everything.
>
> Here is my protocol
> 1) Block in 3% H2O2 for 10'. Wash.
> 2) Block in 10% donkey serum for 1 hr. Wash.
> 3) Block in avidin 15', wash, biotin 15', wash (Vector Labs blocking kits)
> 4) Incubate with primary / isotype overnight at 4C in 2% donkey serum.
> Wash. 5) Incubate with secondary (biotinylated donkey anti-goat, cross
> adsorbed to mouse) in 2% donkey serum for 1 hr at room temp. Wash.
> 6) Incubate with strepavidin HRP in TBS-T for 30'. Wash.
> 7) Incubate with DAB+ (Dako) for 5'.
>
> For isotype, I am using Chrompure Goat IgG, whole molecule from Jackson.
> Some people have suggested that I just do away with isotypes altogether
> and use a no primary control instead. I think there is some merit to this
> idea, but I still think my issue might be indicative of a larger
> technical problem in my staining protocol.
>
> Thanks,
> Adam
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Merced M Leiker
Research Technician II
Cardiovascular Medicine
348 Biomedical Research Building
State University of New York at Buffalo
3435 Main St, Buffalo, NY 14214  USA
leiker <@t> buffalo.edu
716-829-6118 (Ph)
716-829-2665 (Fx)

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