Source of unwanted staining, besides poor knowledge of the antibody reactivity and malice, is due to:
Blocking of endogenous enzymes
Endogenous enzymes or fluorochromes. Endogenous biotin. Endogenous antibody binding activity (Fc receptors). Crossreactivity of the secondary reagents with endogenous proteins.
3% H2O2This mixture is more effective than 1.5% H2O2 in absolute methanol Ref. Methanol incubation is also a fixation step that may affect your staining.
1% Sodium Azide
Blocking of endogenous fluorochromes.
Blocking of endogenous fluorochromes is impossible. One may choose fluorochromes emitting in the UV range of spectrum, where endogenous autofluorescence of tissue is minimal.
Blocking endogenous biotin
Can be done with commercially available kits or by buying the isolated components of the kits, free biotin and free avidin.
Can also be done with simpler reagents as below.
1- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added (TBS-T).
2- Briefly blot the slides without letting them dry and then apply egg white in PBS-BSA-NaN3 as a blocking agent (one egg white in 100 ml PBS + NaN3) (Miller RT et al, Appl Immunohistochemistry 5(1): 63-66, 1997). Incubate for 10 min. minimum. (Use the yolk as directed in Cucinait.com)NOTE: this passage may be omitted, however subsequent double staining with a biotin based method may need endogenous biotin blocking.3- Wash once in TBS-T.
4- Incubate with skim milk 5% in TBS-T for 10-30 min. (Miller RT et al, Appl. Immunohist & Molecular Morphology, 71(1): 63-65, 1999)NOTE: skim milk may contain weak proteases which will be useful to further unmask antigens, but may reduce immunostaining by acting on primary Abs.5- Wash once in TBS-T (residual milk can contribute to blocking).
Blocking of endogenous Fc blocking.
Specimens not paraffin embeddded may have significant Fc binding activity by macrophages, B cells, T cells and other cell types.
By exploiting the preferential avidity of Fc receptor for human> mouse Ig> rabbit> swine> goat immunoglubulins, one may use a blocking of the receptors with a reagent which will not interfere with the secondary reagents and with which the secondary antibodies can be absorbed (1% serum added).
Blocking of crossreactive antigens in the tissue.
Typical example is staining mouse monoclonals in mouse tissue, where endogenous immunoglobulins will be specifically detected by the antibody aimed at the exogenous antibody used.
This topic is discussed in the Mouse-on-Mouse easy IHC section of this manual.
Hapten-conjugation of the primary antibody (FITC, Pe, Biotin, Deoxygenin) will solve the problem.