Introduction:

Antigen detection with mouse primary antibody on mouse tissues is complicated by high levels of background staining when indirect immunohistochemical detection methods are used. The main cause of the background staining is due to the binding of secondary anti-mouse antibody to endogenous mouse tissue Igs and other components. Blocking this binding by pre-incubation with Fab Fragment of Unconjugated Anti-Mouse IgG in combination with the use of a biotin-conjugated Fab Fragment Anti-Mouse IgG (in place of whole labeled secondary antibody) led to the most complete elimination of background staining and achieved satisfactory result. This blocking method can be also adapted to the use of other antibodies on homologous tissues.

This problem often occurs in the following tissue types: muscle, skin and kidney.

Procedure:

1. Paraffin or frozen sections to water.

2. Pretreatment: Perform antigen retrieval or enzyme digestion if needed.

3. Wash 2x2 minutes with PBS-Tween 20.

4. Cleaning: Incubate sections with 1% Triton X-100 diluted in PBS for 30 minutes at room temperature. This step will have a limited reduction in background staining (longer incubation may be more effective, especially for sections thicker than 10 um).

5. Normal Serum Blocking: Without washing, incubate sections directly with normal goat serum blocking solution for 30 minutes at room temperature. Note: Normal serum should be the same species of which the secondary antibody is raised.
6. Wash 3x2 minutes with PBS-Tween 20.
7. Endogenous Mouse IgG Blocking: Incubate sections with Unconjugated AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, Code Number: 115-007-003) for 1 hour at room temperature. Note: (1) dilute the antibody with PBS and make sure its final concentration is NOT lower than 0.1 mg/ml or 100 ug/ml; (2) the antibody concentration lower than 0.1 mg/ml starts to produce background staining; (3) concentration higher than 0.1 mg/ml may be more effective but may not be necessary. 1:10 (0.12 mg/ml) works well for this antibody; (4) increasing blocking time to 2 hours at room temperature or overnight at 4 ºC may be more effective. In this case, lower concentration maybe used.
8. Wash 3x2 minutes with PBS-Tween 20.
9. Primary Antibody: Incubate sections with primary antibody at its optimal dilution in primary antibody dilution buffer for 30 minutes at room temperature.
10. Wash 2x2 minutes with PBS-Tween 20.
11. Endogenous Peroxidase Blocking: Incubate sections with 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase.
12. Wash 3x2 minutes with PBS-Tween 20.
13. Secondary Antibody: Incubate sections with Biotin-SP-conjugated AffiniPure Fab Fragment Goat Anti-Mouse IgG (H+L) (Jackson ImmunoResearch Labs, Code Number: 115-067-003, 1:500 dilution) for 20 minutes at room temperature. Note: (1) longer incubation time may produce more background staining; (2) using whole IgG secondary antibody is not recommended since it may bind to Fc receptors presented in some tissue types.
14. Wash 3x2 minutes with PBS-Tween 20
15. Detection: Incubate sections with HRP-Streptavidin in PBS for 20  minutes at room temperature.
16. Wash 3x2 minutes with PBS-Tween 20
17. Chromogen Substrate: Incubate sections with DAB for 5 minutes.
18. Wash with tap water briefly.
19. Counterstain with hematoxylin as desired.
20. Rinse with tap water.
21. Dehydrate through 95% and 100% ethanol.
22. Clear with xylene.
23. Coverslip with permanent mounting medium.

Images:

Notes:

1. Use TBS-Tween 20 as washing buffer may be more effective (lower background) than using PBS-Tween 20

2. In most cases, background staining is not caused by a single factor when a homologous antibody is used. For example, endogenous peroxidase activity and the presence of endogenous tissue biotin and Igs all played some part of roles here. Block endogenous biotin using avidin/biotin blocking buffer if necessary and this can be done prior to primary antibody incubation.

3. This protocol uses Fab Fragment Goat Anti-Mouse IgG (H+L) to avoid unspecific binding of secondary antibody to Fc receptors presented in mouse tissue, therefore eliminating the need to perform extra blocking step to block endogenous Fc receptors.

4. One can also use conjugated (AP, HRP, FITC, etc.) mouse primary antibody on mouse tissue and this is probably the simplest way to avoid background staining, but sensitivity may be lower. So it may require higher concentration of antibody. 

References:

1. Qi L. Lu and Terry A. Partridge (1998) A New Blocking Method for Application of Murine Monoclonal Antibody to Mouse Tissue Sections. Journal of Histochemistry and Cytochemistry, Vol. 46, 977-984

2. Brown JK, Pemberton AD, Wright SH, Miller HR (2004) Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques. J Histochem Cytochem. 2004 Sep;52(9):1219-30. PubMed Abstract

3. Hierck BP, Iperen LV, Gittenberger-De Groot AC, Poelmann RE (1994) Modified indirect immunodetection allows study of murine tissue with mouse monoclonal antibodies. J Histochem Cytochem. 42(11):1499-502. PubMed Abstract

4. Nielsen B, Borup-Christensen P, Erb K, Jensenius JC, Husby S (1987) A method for the blocking of endogenous immunoglobulin on frozen tissue sections in the screening of human hybridoma antibody in culture supernatants. Hybridoma. 6(1):103-9. PubMed Abstract