Polymer-Based Two-Step IHC Detection
Materials Required
Principle
Polymer-based two-step IHC detects tissue antigens by first binding an unlabeled primary antibody to the antigen and then applying an HRP-polymer secondary reagent that carries multiple secondary antibodies and HRP molecules on a polymer backbone; the localized HRP converts chromogens such as DAB or AEC into visible deposits for light-microscopic interpretation. The method is \"two-step\" because the primary antibody step is followed directly by the polymer-enzyme secondary reagent, rather than by separate secondary-antibody and avidin-biotin complex steps; published comparisons reported similar or higher sensitivity than several multistep systems and avoidance of endogenous-biotin interference.
MCE has not independently verified the accuracy of these methods. They are for reference only.
Experimental Materials
• Use FFPE or frozen tissue sections, antigen-retrieval solution when required for FFPE sections, hydrogen peroxide for endogenous peroxidase blocking in HRP-based detection, wash buffer between incubations, chromogen substrate such as DAB or AEC, hematoxylin counterstain, and mounting medium compatible with the chromogen used.
• Use a primary antibody validated for IHC on the intended tissue preparation, an HRP-polymer secondary detection reagent directed against the host species of the primary antibody, and positive and negative control slides processed with the same staining workflow.
• Use a microtome or cryostat for section preparation, staining jars or an automated immunostainer, a humidity chamber or controlled-temperature incubator for antibody incubations, heating equipment for antigen retrieval when FFPE antigen retrieval is required, and a bright-field microscope for chromogenic readout.
• Use a primary antibody validated for IHC on the intended tissue preparation, an HRP-polymer secondary detection reagent directed against the host species of the primary antibody, and positive and negative control slides processed with the same staining workflow.
• Use a microtome or cryostat for section preparation, staining jars or an automated immunostainer, a humidity chamber or controlled-temperature incubator for antibody incubations, heating equipment for antigen retrieval when FFPE antigen retrieval is required, and a bright-field microscope for chromogenic readout.
Experimental Procedure
• Prepare tissue sections according to the specimen type: FFPE sections require deparaffinization and hydration before staining, whereas frozen-section rapid protocols in the EnVision study used acetone fixation for 1 min followed by air-drying before antibody incubation.
• For FFPE tissues, apply antigen retrieval only when required for the antibody-antigen pair;
• Antigen retrieval is a high-temperature pretreatment used to improve immunostaining of formalin-fixed paraffin-embedded tissues, but retrieval conditions should be optimized rather than assumed universal.
• Perform staining in this order: block endogenous peroxidase for HRP-based detection;
• Incubate with the primary antibody;
• Wash;
• Incubate with the HRP-polymer secondary reagent;
• Wash;
• Develop with DAB or AEC until visible signal is generated;
• Counterstain;
• And mount for bright-field microscopy.
• Reported incubation parameters vary by tissue and workflow: the rapid frozen-section EnVision protocol used 3 min each for primary antibody, EnVision complex, and chromogen at 37°C, whereas FFPE polymer-based diagnostic studies used antigen retrieval and non-biotin polymerized HRP detection in formalin-fixed paraffin-embedded tissues without establishing a single universal incubation time for all antibodies.
• Interpret positive staining as antigen-localized chromogen deposition in the expected cellular or tissue compartment, and compare each run with a known positive tissue control and a negative control processed without specific primary antibody or with an irrelevant antibody when reported by the staining design.
• For quantitative or semi-quantitative use, record staining intensity, percentage of expected positive cells, localization pattern, and batch conditions;
• Comparative EnVision studies used antibody titration and comparison across detection systems to judge sensitivity and reproducibility.
• For FFPE tissues, apply antigen retrieval only when required for the antibody-antigen pair;
• Antigen retrieval is a high-temperature pretreatment used to improve immunostaining of formalin-fixed paraffin-embedded tissues, but retrieval conditions should be optimized rather than assumed universal.
• Perform staining in this order: block endogenous peroxidase for HRP-based detection;
• Incubate with the primary antibody;
• Wash;
• Incubate with the HRP-polymer secondary reagent;
• Wash;
• Develop with DAB or AEC until visible signal is generated;
• Counterstain;
• And mount for bright-field microscopy.
• Reported incubation parameters vary by tissue and workflow: the rapid frozen-section EnVision protocol used 3 min each for primary antibody, EnVision complex, and chromogen at 37°C, whereas FFPE polymer-based diagnostic studies used antigen retrieval and non-biotin polymerized HRP detection in formalin-fixed paraffin-embedded tissues without establishing a single universal incubation time for all antibodies.
• Interpret positive staining as antigen-localized chromogen deposition in the expected cellular or tissue compartment, and compare each run with a known positive tissue control and a negative control processed without specific primary antibody or with an irrelevant antibody when reported by the staining design.
• For quantitative or semi-quantitative use, record staining intensity, percentage of expected positive cells, localization pattern, and batch conditions;
• Comparative EnVision studies used antibody titration and comparison across detection systems to judge sensitivity and reproducibility.
Troubleshooting
Weak or absent staining
May result from insufficient antigen accessibility or inadequate detection sensitivityOptimize antigen retrieval for FFPE tissues and perform antibody titration because antigen retrieval and detection-system choice affected staining intensity and usable antibody dilution in comparative IHC studies.
False-positive or diffuse background staining
May result from endogenous peroxidase activity or detection-system interferenceInclude endogenous peroxidase blocking and negative controls, and use non-biotin HRP-polymer detection when endogenous biotin interference is a concern.
Poor performance in archival FFPE tissue
May result from fixation or long-term storage effects reducing antigen detectabilityUse antigen retrieval and validate the polymer-HRP method on appropriate positive archival material before applying it diagnostically.
Referencias:
- [1]. Sabattini E, et al. The EnVision++ system: a new immunohistochemical method for diagnostics and research. Critical comparison with the APAAP, ChemMate, CSA, LABC, and SABC techniques. J Clin Pathol. 1998;51(7):506-511. [Content Brief]
- [2]. Kämmerer U, et al. A new rapid immunohistochemical staining technique using the EnVision antibody complex. J Histochem Cytochem. 2001;49(5):623-630. [Content Brief]
- [3]. Shi SR, et al. Sensitivity and detection efficiency of a novel two-step detection system (PowerVision) for immunohistochemistry. Appl Immunohistochem Mol Morphol. 1999;7(3):201-208.
- [4]. Shi SR, et al. Antigen retrieval immunohistochemistry: past, present, and future. J Histochem Cytochem. 1997;45(3):327-343. [Content Brief]
- [5]. Liang CT, et al. A non-biotin polymerized horseradish-peroxidase method for the immunohistochemical diagnosis of canine distemper. J Comp Pathol. 2007;136(1):57-64. [Content Brief]
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