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Blocking Steps

Consideration of what may already be in the tissue can improve the staining results.

Background staining in immunohistochemistry can be very frustrating. At best it makes sections murky, but readable, at worst it can obscure the desired staining completely, or give misleading results.  As immunohistochemistry relies on a number of natural molecules for the detection system, there is a chance that these can be found within the tissue and cause problems. 

Ig receptors

Cells that are involved in the immune response may be able to bind to antibodies used in the detection process.  Although there is some debate over the ability of these receptors to bind Ig from different species, it is customary to include a source of Ig/serum proteins from the host species of any secondary antibody to be used. Any Ig receptors in the tissue should bind the Ig from the serum, rather than non-specifically binding the primary antibody. When the secondary antibody is applied, it's very unlikely to inadvertently recognise Ig from the species in which it was raised - this might only be expected in some auto-immune conditions.

BSA is often used as a ubiquitous source of Ig for blocking, however if the primary antibody is made from a bovine, caprine or ovine source this may increase background!

In cases where it is determined that this is a cause of background, even with blocking, using Ig fragments (Fab'), rather than whole antibodies can improve the staining. 

Protein Blocking


The levels of endogenous Ig can vary widely in tissues, depending on the immune status of the tissue donor being tested, and also whether or not the system is being challenged. In most cases, this issue can be avoided by careful selection of the species involved - avoiding closely related species in the detection systems, however there is a strong predominance of primary antibodies made in mouse or rat and these are also a well used test subjects! Choosing adsorbed secondary antibodies can help in cases such as mouse primary antibodies on rat tissues and vice versa. Where the test primary antibody is of the same species as the tissue, specialist Species-on-Species kits may be required. 

Species On Species Kits

Endogenous enzymes

Horseradish peroxidase and alkaline phosphatase are the most commonly used enzymes in detection systems. They generally give good sensitivity, and their substrates deposit discretely giving crisp staining. Sometimes the amount of endogenous enzyme present in the tissue can make one of these preferential over the other. 

Peroxidase Blocking

A hydrogen peroxide block can be used, although a this harsh blocking step may cause fizzing on the slide, and damage the tissue. The best method may also depend on the type of tissue and embedding it was subjected to. Some of these methods may also be useful when multiple labelling/ multiplexing when a first label using HRP needs to be inactivated.

Peroxidase Blocking Methods         RTU Peroxidase Blocking Solutions

AP Blocking 

Alkaline phosphatase comes naturally in different isoforms. In many tissues, the isoform is one that can be inhibited by levamisole (1mM), added to the substrate working solution, however the intestinal isoform is not blocked in this manner. Detection kits utilise this fact, and will contain the intestinal form of the enzyme. In the gut therefore, any endogenous AP must be blocked before staining by other methods, such as 1% acetic acid, or 0.3M hydrochloric acid, but these methods may also damage antigens, so peroxidase may become a better choice of reporting enzyme in these tissues.

RTU AP Blocking Solutions


In methods using biotinylated reagents, any endogenous biotin should be blocked at the start of the proceedure. Biotin levels vary by organ type with liver and kidney having notoriously high levels. Blocking is simple in most cases - sections are incubated with avidin or streptavidin, dependent on the detection reagent, which binds the endogenous biotin. As the (strept)avidin have multiple biotin binding sites, these are then filled by an incubation with free biotin. Biotin can only bind to one (strept)avidin binding site, so this effectively blocks these reagents preventing further interactions. To reduce the protocol time, sometimes it is possible to include this block into other reagents in the normal protocol - adding the (strept)avidin to the normal serum block, and the biotin into the primary antibody solution.  This short cut is not recommended for biotinylated primary antibodies. 

Biotin Blocking Solutions