Immunohistochemistry Resource Guide

Table of Contents

Introduction

Immunohistochemistry Workflow

Immunohistochemistry Selection Guide

Pioneering in IHC Technology

Choosing a Detection System

Avidin-Biotin Complex (ABC)-Based Detection

VECTASTAIN® ABC Systems

VECTASTAIN® ABC Kits

Choosing a VECTASTAIN® ABC Kit

> Customizing your VECTASTAIN® ABC Kit

Polymer-Based Detection

ImmPRESS® One-Step Polymer Systems

> ImmPRESS® Two-Step Amplified Polymer Systems

> Choosing an ImmPRESS® Polymer Kit

> Multiple Antigen Labeling Simplified

Species on Species Detection

M.O.M.® (Mouse on Mouse) Immunodetection Kits

Choosing an Enzyme Substrate

Enzyme Substrates

> Enzyme Substrate Properties

Multiple Antigen Labeling

Enzyme Substrate Combinations

Counterstaining

> Counterstain/Substrate Compatibility Table

Blocking Background Signal

Secondary and Tertiary Detection Reagents

Mounting Media

Accessory Reagents

Choosing a Detection System

Immunohistochemistry Overview

Immunohistochemistry (IHC) is a method to detect specific target antigens (proteins) in tissue sections using antibodies. Immunocytochemistry (ICC) uses similar techniques to localize cellular proteins in cell preparations. Both IHC and ICC are powerful tools that provide insights into gene expression, spatial relationships, and biomarker identification in a wide variety of applications. These applications include basic research, assessment of normal and disease states within human and animal tissues, and assessment of plant pathology.

The target antigen, bound by the detection antibody, is visualized using either chromogenic or fluorescence detection. In chromogenic detection, the detection antibody is conjugated to an enzyme. The enzyme, usually horseradish peroxidase or alkaline phosphatase, catalyzes the conversion of its respective chromogen to a colored precipitate at the site of the antigen. This precipitate can be visualized by using brightfield microscopy. Certain chromogens can also be visualized by using electron, darkfield or fluorescence microscopy. In fluorescence detection, the detection antibody is conjugated to a fluorophore which can be visualized using fluorescence microscopy.

For the purposes of this antibody selection guide, IHC will be referenced for both IHC and ICC techniques.

Comparison of Detection Systems and Antibody Selection

Choose the appropriate detection system for your experiment based on enzyme, sensitivity, cost, biotin vs. non-biotin formats, flexibility, and time considerations. These parameters will help you with your antibody selection.

Detection System	Enzyme	Sensitivity	Cost/ Assay	Biotin- Free	Micro- polymer	Modular	Mouse Primary on Mouse Tissue	Ready-to- Use (R.T.U.) Format	Typical number of steps
ImmPRESS® Kits
ImmPRESS® Excel Amplified HRP Polymer Kits	HRP	••••••	••••	•	•			•	2
ImmPRESS® HRP Polymer Kits	HRP	•••••	•••	•	•			•	1
ImmPRESS® VR HRP Polymer Kits	HRP	•••••	••••	•	•			•	1
ImmPRESS® AP Polymer Kits	AP	•••••	••••	•	•			•	1
VECTASTAIN® Kits
VECTASTAIN® Elite® ABC Kits	HRP	•••••	••			•			2
R.T.U. VECTASTAIN® Elite® Kits	HRP	•••••	••			•		•	2
VECTASTAIN® Universal Quick Kits	HRP	••••	••			•			2
R.T.U. VECTASTAIN® Universal Quick Kits	HRP	••••	••			•		•	2
VECTASTAIN® ABC-AP Kits	AP	••••	•			•			2
Original VECTASTAIN® ABC Kits	HRP	•••	•			•			2
M.O.M.® (Mouse on Mouse) Kits
M.O.M.® (Mouse on Mouse) ImmPRESS® Polymer Kit	HRP	•••	•••	•	•		•		1
M.O.M.® (Mouse on Mouse) Kits	HRP	•••	•••			•	•		2
Additional Options
Enzyme Conjugated Avidin/Streptavidin	HRP or AP	•••	•			•			2
R.T.U. HRP Avidin/Streptavidin	HRP	•••	•			•		•	2
Enzyme Conjugated Secondary Antibody	HRP or AP	••	•	•					1

HRP – Horseradish peroxidase
AP – Alkaline phosphatase
VR – Veterinary Reagents

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