Hi kumari

Brown/Red color observed on Negative Control Tissue:

- Monoclonal antibody: Possible contamination.

-Polyclonal antibody: Possible contamination or undesired antibody

in the host Ig fraction.

-

-Antigen retrieval lipofusion-artifact may appear as granule staining

in liver and cardiac tissue, or as specific staining in pancreatic

sections

 the brown/red color Indicates endogenous peroxidase activity in the tissue sections. so what you can do It is  to block with three percent hydrogen peroxide or other peroxidase blocking reagent. Using a new bottle of hydrogen peroxide, perform a three percent H202 peroxidase block, followed by DAB and an appropriate counterstain

Hi,

Did you incubate the sections in species specific serum. As mentioned above you should also include quenching the sections with 3% H2O2 in buffer you use for IHC washing. Are you using paraffin sections or those are cryosections?

If the positivity appears as single positive cells resembling plasma cells and you are using an indirect protocol with secondary biotinylated antibodies, it will probably be about the secondary antibody, which under some circumstances (depending on the antigen retrieval method, for example) can attach unspecifically to plasma cells. It happened to me and I solved this problem by adding to the secondary antibody 2% of normal serum of the same species of the tissue (In my case I work with bovine tissues, so I added 2% of normal bovine serum) and it worked greatly (normal serum immunoglobulins block the Fc receptors of plasma cells that unspecifically attrack the secondary antibodies). I hope it helps!

The color you observed in negative control (in the absence of your primary and secondary antibodies) is due certainly to endogenous peroxidase as mentioned in most responses. In general, it's easy to eliminate or at least  to diminish this back ground by using 3% H2O2 or methanol at RT. You can also improve this diminution by using a preincubation step (PBS with Albumin Bovine Serum ans Normal Serum) to mask undesirable antigens. 

Which detection-system do you use? LSAB? polymer? ABC? For the systems, that use streptavidin you have to block the endogenous biotin. This is often found in liver, gut and kidney; but after antigen retrieval it may be seen in several tissuetypes.

Does it look like "real IHC signal" or just like unspecific staining?

Thanks to all for their valuable suggestion.

I use monoclonal antibody on paraffin embedded sections of synovial tissue. I block the endogenous peroxidase activity with 3% H2O2 for nearly more than an hour. For blocking I use serum of the species in which secondary is raised (like if secondary is generated in rabbit then I use rabbit serum for blocking). Also, I used to boil the tissue section in citrate buffer (pH 6.0) for around 20 minutes.

I observed that almost all the cells present in the vessels of tissue is completely brown in color.

How old is your 3% H2O2. The cells in the vessels are presumely erythrocytes. And if ery show DAB-staining the blocking of endogenous peroxidase is insufficient.

You can proof this by omitting every step of IHC protocol after AR until DAB-development. If you see the same result as in your test-section, then it is definitly insufficient.

This can result from any of a number of possible situations where peroxidase activity is present in the tissue in a manner not consistent with your antigen's distribution. There can be a number of causes which can be diagnoses and fixed (unless the antigen is also destroyed).

1) endogenous peroxidase activity; test: tissue without peroxidase added; cure: kill the peroxidase activity as has been already described

2) tissue binding of a component of your signalling mechanism (primary antibody, secondary antibody, other components); test: apply components of your signalling mechanism starting from the peroxidase and working back toward the primary antibody. This will reveal non-specific binding of these components to the tissue; cure: block the binding with a presoak of serum from that species (or an unlabeled component of non-immune molecules).

I have found amphioxus tissue that bound peroxidase. I could block this binding by presoaking with a presoak of peroxidase with the heme (and therefore the enzymatic activity) removed.

Generally, non-specific binding of antibodies is not uncommon, but can be eliminated with a presoak fo the appropriate series serum.

I have found that non-specific labeling by DAB solution can sometimes be reduced or eliminated by pushing the unreacted DAB solution through 1 micron syringe filter.

The main quantity of endogenous peroxidase is in neutrophyil granulocytes. It's depend on what tissue you are studing.

Other artefacts can be due to

 1 drying sections 

2 primary or secondary antibody crossreaction with sections proteins and molecules.

that have to be blocked with BSA or other sera.

All the above answers are right the important cause of non-specific staining are:

1. Endogenous peroxidase in RBC and macrophages- so go for blocking of endogenous peroxidase after antigen retrieval for this we can 3% H2O2 in methanol for 15-30 minutes before protein blocking.

2. Non-specific binding of primary antibody. To avoid this go for  protein blocking before adding of primary antibody for 15-30 minutes. Use 3-5% BSA or commercially available protein blocking solutions such as power block of Biogenex Company.

3. Use of highly concentrated primary antibodies especially polyclonal antibody. So go for dilution of primary antibody in washing buffer and BSA. 

Increased background staining can also occur if the concentration of H2O2 in the DAB solution is too high.

• All of the above are valid.  I would add endogenous biotin if you are using a biotin based detection.   Kidney and liver contain the most biotin but it can be seen in other tisis This can be eliminated by blocking biotin with the appropriate blocking kit or reagents by but they 

Using H2O2 is the best way we used to prevent elevated background resulting from endogenous peroxidase in erythrocytes. Depending on your tissue type, you have to experiment different concentrations and/or different times of H2O2 application. Also, as said by Tony Henwood, be careful to the concentration of H2O2 in the final solution of DAB to reveal the immunostaining. I would just add at this step to control visually the product of the reaction to avoid high background. Time of revelation is also crucial.

Hi, I support all other points. I am doing IHC on a day to day basis. I think the problem would be the negative control tissue itself. To rule out the background issue you can run the negative control without your target antibody (just use PBS or saline) and compare with your Antibody treated slides. Are you using any blueing solutions after Heamatoxylin step ?. Antigen retreaval is another area that you can consider. If you cook the tissue too much (via oven or pressure cooker) it can cause non specific staining in negative control tissues. Good Luck

Hi,

first of all I carry out negative controls, along with internal and positive controls, at the same time to  "standardize" the protocol. After that, you may try to perform additional negative controls, using secondary Abs from different species to determine if the positivity arises from crosslinking of the secondary Ab. Sometime I face this using biotinilated anti-goat Abs.

Additionally, it could be useful an internal control, i.e. using Cytokeratins or Vimentin, to be sure the false positivity it is not caused by a weak blocking of endogenous peroxidase activity or negative charged proteins in the background.

Hope to be useful