[Histonet] Isotype background

[Andrea T. Hooper]

Your protocol is fine. It's precisely my protocol.  


What do you get when you do the no primary control? Although this control isn't sufficient for publication it is necessary for troubleshooting. In fact performing the following controls together can tell you exactly where the problem lies:

(1) IgG control - tells you if you are getting non-specific binding due to interactions of primary with the tissue itself or with the reagents in the protocol
(2) No primary, with secondary, with strepavidin, with DAB - tells you if the secondary is giving background
(3) No primary, no secondary, with streptavidin, with DAB - tells you endogenous biotin background
(4) No primary, no secondary, no streptavidin, with DAB - tells you endogenous peroxidase background
(5) Blank - Nothing at all - just take it through staining elimination ALL reactive steps including DAB - tells you if you have endogenous pigment or hemosiderin etc in your section

(6) The most important control in any experiment is the negative tissue control. This means perform your experiment specifically on tissue in which you know does not express your protein of interest. Without this control you cannot be sure what you are seeing is specific (interpreted as either your protocol has issues or the primary you are working with detects other related proteins etc).

That being said, my personal experience with Chrompure IgGs is that over time they get "dirty". I am not sure whether this is due to a contamination from overuse (they sure do provide a huge amount of reagent in each vial!!) or degradation from another cause. I think it's contamination as when I aliquot them out, I get it much less than before. Also be sure you are diluting it appropriately as the stock is 11mg/ml or so and your primaries are probably 10 fold less. Is there another goat IgG you can use as a sort of "positive control". An antibody you know will stain in a certain pattern from personal experience - see if it gives you a better pattern as I think your IgG may just be "bad".

You are not using BSA are you? I ask b/c goat secondaries made in donkey may cross react to bovine IgG present in BSA.

Finally are you working with paraffin or frozen?


--- On Tue, 10/13/09, Adam . <anonwums1 <@t> gmail.com> wrote:

From: Adam . <anonwums1 <@t> gmail.com>
Subject: [Histonet] Isotype background
To: histonet <@t> lists.utsouthwestern.edu
Date: Tuesday, October 13, 2009, 9:48 PM

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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