The indirect detection of mouse primary antibodies on mouse tissues is often complicated by high background staining. The major problem is the inability of anti-mouse secondary antibody to distinguish between endogenous mouse immunoglobulins in the tissue and mouse the primary antibody.
Endogenous immunoglobulins are mainly distributed in intercellular space in soluble form and will cause particular problems for the detection of antigens localized near cell surface or in extracellular matrix. They can also be found on the cell surface of lymphocytes.
The level of background is tissue type-related and varies from sample to sample. The problem often occurs in tissues of muscle, skin and kidney.
Additionally endogenous peroxidase activity in the sample tissue or Fc-Receptor interaction with the detection antibodies can also be a cause for high background.
Peroxidase activity is inhibited by a blocking step before the incubation with normal serum or after the primary antibody incubation.
Binding of secondary antibody to Fc receptors on cells and tissues can often be inhibited by a suitable blocking with normal serum or with special Fc receptor blocking solutions.
The most efficient way to prevent the secondary antibody from binding is the use of F(ab’)2 fragment secondary antibodies.
Blocking of endogenous immunoglobulins using monovalent Fab-fragments
A particularly efficient method to prevent the background staining of endogenous immunglobulins in indirect detection methods is the pre-incubation of the tissue sample with Fab-fragments of unconjugated anti-mouse antibodies. This method can also easily be adapted to the indirect detection (Immunohistochemistry, Immunofluorescence, Cytometry) of primary antibodies from other species on homologous tissues and cells.
Example protocol for blocking with monovalent Fab-Fragments.
Monovalent Fab-Fragments for blocking
Fab anti-Mouse
Fab anti-Rat
Fab anti-Human
Fab anti-Rabbit
IHC Antibodies – Mouse Histology
IHC-validated marker for Mouse Histology – Specifically developed for murine formalin-fixed and paraffin-embedded tissue (FFPE)! Antibodies for Mouse Histology
Immunohistochemical staining of mouse antibodies on rat tissue
The detection of mouse antibodies on tissues of homologous species like rat impose special requirements to the secondary detection reagents.
Due to the high homology between mouse and rat secondary antibodies against mouse IgG will cross react with rat IgG and can cause background staining of endogenous rat immunoglobulins.
Soluble endogenous immunoglobulins are mainly located in intercellular space and can cause particular problems with the detection of cell surface antigens and those located in the extracellular matrix.
The level of background depends on tissue type and the individual sample preparation, but often occurs in muscle, skin and kidney.
One method for the inhibition of background staining of endogenous immunoglobulins using indirect detection is described here (mouse antibodies on mouse tissue).
A simpler and more efficient method for avoiding background staining is the usage of secondary antibodies with minimized cross reactivity (MinX) to the species of the target tissue (i.e. rat).
For the staining of rat tissue follow the example protocol without step 7./8. and use e. g. cat.# 115-035-166 in step 11.
Note: Due to the adsorption against a closely related species monoclonal primary antibodies with rare isotypes (IgG2b, IgG3) may not be recognized optimally.
The sensitivity of a secondary antibody adsorbed against Ig of a homologous species has a reduced sensitivity. This is especially true for anti-mouse (adsorbed against rat), anti-rat (adsorbed against mouse) or anti-(arm.)hamster (adsorbed against mouse, rat).
It should therfore be avoided to choose a secondary antibody adsorbed against a closely related species, if it is not necessary.