Avidin-Biotin/Streptavidin-Biotin IHC
Materials Required
Principle
Avidin-biotin or streptavidin-biotin immunohistochemistry detects tissue antigens by binding a primary antibody to the target antigen, then detecting that antibody with a biotinylated antibody and an avidin-biotin-enzyme or streptavidin-enzyme detection complex; the enzyme reaction produces a visible chromogenic deposit at the antigen site for light-microscopic localization[1][2][3]. The classic ABC method uses the high-affinity avidin-biotin interaction to bridge biotinylated secondary antibody and biotinylated peroxidase, and early comparative studies reported stronger immunoperoxidase staining than PAP-based methods in formalin-fixed tissue sections[2][3].
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use fixed tissue sections, wash buffer, blocking reagents for nonspecific protein binding, hydrogen peroxide when HRP-based peroxidase detection is used, chromogenic substrate compatible with the enzyme label, counterstain, dehydration reagents, clearing reagent, and mounting medium; these materials support tissue preparation, reduction of background signal, enzyme-mediated color development, nuclear or tissue contrast, and slide preservation[4][10].
• Use a target-specific primary antibody, a biotinylated secondary antibody directed against the primary antibody species, and either an avidin-biotin-peroxidase complex or a streptavidin-enzyme conjugate; the essential detection logic is primary antibody binding, biotinylated secondary antibody binding, and avidin/streptavidin-based enzyme localization[1][2][4].
• Use a microtome or cryostat for sectioning, microscope slides, humidified incubation chamber, staining jars or automated stainer, heat source for antigen retrieval when validated for the antigen, and a bright-field microscope for chromogenic readout evaluation[4][5][10].
• Use a target-specific primary antibody, a biotinylated secondary antibody directed against the primary antibody species, and either an avidin-biotin-peroxidase complex or a streptavidin-enzyme conjugate; the essential detection logic is primary antibody binding, biotinylated secondary antibody binding, and avidin/streptavidin-based enzyme localization[1][2][4].
• Use a microtome or cryostat for sectioning, microscope slides, humidified incubation chamber, staining jars or automated stainer, heat source for antigen retrieval when validated for the antigen, and a bright-field microscope for chromogenic readout evaluation[4][5][10].
Experimental Procedure
• Prepare fixed paraffin or frozen tissue sections using a tissue-processing workflow appropriate for the specimen and antigen, because fixation, tissue processing, section preparation, and antigen retrieval can affect IHC staining intensity and interpretability[4][5][10].
• For formalin-fixed paraffin-embedded sections, include deparaffinization, rehydration, optional antigen retrieval, blocking steps, antibody incubation, detection, chromogen development, counterstaining, dehydration, clearing, and mounting as the overall workflow; antigen retrieval should be used only after optimization because heating can improve antigen detection but can also expose endogenous biotin-like activity in some tissues[4][5][8].
• Prepare controls before staining: a known positive tissue or cell control for the target antigen, a negative reagent control such as primary-antibody omission or matched irrelevant antibody control, and an endogenous-biotin control when using avidin-biotin or streptavidin-biotin detection on tissues prone to biotin-related artifact[6][7][8][10].
• Section, mount, deparaffinize, and rehydrate FFPE tissue sections, or prepare fixed frozen sections, using a validated IHC tissue-preparation workflow[4][10].
• Perform antigen retrieval only when it improves the validated antibody-antigen signal for the tissue and target; heat-based retrieval has been widely used to enhance staining in FFPE sections, but it may increase endogenous biotin-related signal and therefore requires appropriate controls in biotin-based detection systems[5][8].
• Block endogenous peroxidase activity before HRP-based detection when tissue or blood elements may generate peroxidase-related background[10].
• When endogenous avidin-binding or endogenous biotin activity is possible, apply an avidin/biotin blocking strategy before the biotinylated antibody and avidin/streptavidin detection steps; published work showed endogenous avidin-binding activity could be detected and suppressed in selected human and murine tissues[6][7][8].
• Incubate sections with the target-specific primary antibody under an antibody-validated condition, wash, incubate with a biotinylated secondary antibody, wash, then incubate with avidin-biotin-peroxidase complex or streptavidin-enzyme reagent[1][2][4].
• Apply the enzyme substrate until specific chromogenic signal is visible under controlled conditions, stop the reaction, counterstain if needed, dehydrate or otherwise process according to the chromogen and mounting system, mount, and examine by bright-field microscopy[2][4][10].
• Interpret positive staining by its expected tissue, cellular, and subcellular localization rather than signal presence alone; compare each experimental slide with positive controls, negative reagent controls, and endogenous-biotin controls because endogenous biotin in tissues such as kidney, liver, and brain can mimic true staining in avidin/streptavidin systems[7][8][10].
• Quantification should be treated cautiously because chromogenic IHC is often semiquantitative; image analysis or scoring should use predefined criteria, consistent acquisition settings, and the same validated staining workflow across compared samples[10][11].
• For formalin-fixed paraffin-embedded sections, include deparaffinization, rehydration, optional antigen retrieval, blocking steps, antibody incubation, detection, chromogen development, counterstaining, dehydration, clearing, and mounting as the overall workflow; antigen retrieval should be used only after optimization because heating can improve antigen detection but can also expose endogenous biotin-like activity in some tissues[4][5][8].
• Prepare controls before staining: a known positive tissue or cell control for the target antigen, a negative reagent control such as primary-antibody omission or matched irrelevant antibody control, and an endogenous-biotin control when using avidin-biotin or streptavidin-biotin detection on tissues prone to biotin-related artifact[6][7][8][10].
• Section, mount, deparaffinize, and rehydrate FFPE tissue sections, or prepare fixed frozen sections, using a validated IHC tissue-preparation workflow[4][10].
• Perform antigen retrieval only when it improves the validated antibody-antigen signal for the tissue and target; heat-based retrieval has been widely used to enhance staining in FFPE sections, but it may increase endogenous biotin-related signal and therefore requires appropriate controls in biotin-based detection systems[5][8].
• Block endogenous peroxidase activity before HRP-based detection when tissue or blood elements may generate peroxidase-related background[10].
• When endogenous avidin-binding or endogenous biotin activity is possible, apply an avidin/biotin blocking strategy before the biotinylated antibody and avidin/streptavidin detection steps; published work showed endogenous avidin-binding activity could be detected and suppressed in selected human and murine tissues[6][7][8].
• Incubate sections with the target-specific primary antibody under an antibody-validated condition, wash, incubate with a biotinylated secondary antibody, wash, then incubate with avidin-biotin-peroxidase complex or streptavidin-enzyme reagent[1][2][4].
• Apply the enzyme substrate until specific chromogenic signal is visible under controlled conditions, stop the reaction, counterstain if needed, dehydrate or otherwise process according to the chromogen and mounting system, mount, and examine by bright-field microscopy[2][4][10].
• Interpret positive staining by its expected tissue, cellular, and subcellular localization rather than signal presence alone; compare each experimental slide with positive controls, negative reagent controls, and endogenous-biotin controls because endogenous biotin in tissues such as kidney, liver, and brain can mimic true staining in avidin/streptavidin systems[7][8][10].
• Quantification should be treated cautiously because chromogenic IHC is often semiquantitative; image analysis or scoring should use predefined criteria, consistent acquisition settings, and the same validated staining workflow across compared samples[10][11].
Troubleshooting
Problem: diffuse or tissue-pattern background in a biotin-based IHC run.
• Possible Cause: endogenous biotin or endogenous avidin-binding activity.• Literature-supported Solution: include endogenous-biotin controls and apply avidin/biotin blocking before biotinylated-secondary and avidin/streptavidin detection; if artifact remains, interpret biotin-based staining cautiously, especially after antigen retrieval[6][7][8].
Problem: apparent positive staining in negative controls after heat retrieval.
• Possible Cause: antigen retrieval can expose or enhance endogenous biotin-like activity.• Literature-supported Solution: repeat staining with negative controls processed through the same retrieval conditions and consider a non-biotin detection system when endogenous biotin artifact cannot be resolved[8][9].
Problem: background differs between ABC and polymer-based visualization systems.
• Possible Cause: the detection system itself can contribute to background staining in FFPE tissues.• Literature-supported Solution: compare detection systems during assay validation and use the system that gives acceptable specific staining with lower background for the tissue-antigen pair[9][10].
References:
- [1]. Guesdon JL, et al. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem. 1979;27(8):1131-1139. [Content Brief]
- [2]. Hsu SM, et al. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: a comparison between ABC and unlabeled antibody (PAP) procedures. J Histochem Cytochem. 1981;29(4):577-580. [Content Brief]
- [3]. Hsu SM, et al. The use of antiavidin antibody and avidin-biotin-peroxidase complex in immunoperoxidase technics. Am J Clin Pathol. 1981;75(6):816-821. [Content Brief]
- [4]. Magaki S, et al. An introduction to the performance of immunohistochemistry. Methods Mol Biol. 2019;1897:289-298. [Content Brief]
- [5]. Shi SR, et al. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem. 1991;39(6):741-748. [Content Brief]
- [6]. Wood GS, et al. Suppression of endogenous avidin-binding activity in tissues and its relevance to biotin-avidin detection systems. J Histochem Cytochem. 1981;29(10):1196-1204. [Content Brief]
- [7]. Wang H, et al. Detection of endogenous biotin in various tissues. Histochem Cell Biol. 1999;112(1):77-83. [Content Brief]
- [8]. Bussolati G, et al. Retrieved endogenous biotin: a novel marker and a potential pitfall in diagnostic immunohistochemistry. Histopathology. 1997;31(5):400-407. [Content Brief]
- [9]. Vosse BA, et al. Background staining of visualization systems in immunohistochemistry: comparison of the Avidin-Biotin Complex system and the EnVision+ system. Appl Immunohistochem Mol Morphol. 2007;15(1):103-107. [Content Brief]
- [10]. Ramos-Vara JA. Technical aspects of immunohistochemistry. Vet Pathol. 2005;42(4):405-426. [Content Brief]
- [11]. Taylor CR, et al. Quantification of immunohistochemistry--issues concerning methods, utility and semiquantitative assessment II. Histopathology. 2006;49(4):411-424. [Content Brief]
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