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Immunohistochemistry Resource Guide

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IHC Guide Cover v1 IHC-Guide-Cover_v1 IHC-Guide-Cover_v1

Table of Contents

Choosing a VECTASTAIN® ABC Kit

Choose the detection enzyme

  • Peroxidase
  • Alkaline phosphatase
Choose the appropriate ABC kit

To detect a biotinylated target, you will need:

  • VECTASTAIN ABC Reagent contained in the standard VECTASTAIN ABC Kit
  • An appropriate substrate To detect an unlabeled primary antibody or lectin, you will need:
  • A biotinylated secondary antibody that binds to the primary antibody species or lectin you have chosen
  • VECTASTAIN ABC Reagent
  • An appropriate substrate

For example, to detect a primary antibody made in rabbit, the appropriate choice is a VECTASTAIN ABC Kit designated Rabbit IgG.

Choose a convenient format VECTASTAIN ABC Kits are available in economical concentrated formats. For additional convenience and ease of use some peroxidase-based kits are offered in ready-to-use prediluted stabilized formats.
tumor Ki67 tumor-Ki67 tumor-Ki67
Tumor: • Ki67 (m), VECTASTAIN Elite® ABC Kit, Vector® DAB (brown) • CD34 (m), VECTASTAIN ABC-AP Kit, Vector Blue (blue) • Cytokeratin AE1/AE3 (m), VECTASTAIN ABC-AP Kit, Vector Red (red).

Consider Species Cross-Reactivity

When choosing the optimal detection system for your application, it is important to consider not only the species of the primary antibody but also the species of the tissue under examination. If the species of the primary antibody and the species of the tissue are closely related (for example, rat and mouse), the biotinylated secondary antibody may cross-react with endogenous IgG in the tissue section. This can lead to background staining.

The following options may minimize background staining in these instances:

  • Directly label the primary antibody with biotin (ChromaLINK® One-Shot™ Antibody Biotinylation Kit, B-9007) and detect it using the VECTASTAIN Elite ABC Kit (Standard, PK-6100, includes VECTASTAIN Elite ABC Reagent only) followed by an HRP substrate. Alternatively, label the primary with HRP (SoluLINK™ Protein-Protein Conjugation Kit, S-9010) and develop with an HRP substrate.
  • Use a biotinylated secondary antibody specifically adsorbed to remove cross-reacting antibodies of closely-related species (e.g. biotinylated anti-mouse IgG, rat adsorbed).
  • Use the M.O.M.® (Mouse on Mouse) Immunodetection System for applications of mouse primary antibodies on mouse tissue.

Substrates

After choosing the VECTASTAIN ABC Kit for your application, select a substrate that matches the enzyme system of the kit.

Endometrium substrate brown Endometrium-substrate-brown Endometrium-substrate-brown
Endometrium: • Progesterone Receptor (rm), VECTASTAIN Universal ABC- AP Kit, Vector® Blue AP Substrate (blue) • CD34 (m), VECTASTAIN Universal Elite ABC Kit, Vector DAB HRP Substrate (brown).
Tumor substrate blue gray Tumor-substrate-blue-gray Tumor-substrate-blue-gray
Tumor: • p53 protein (m), VECTASTAIN® ABC-AP Kit, Vector Red AP Substrate (red) • Pan-Cytokeratin (sheep), VECTASTAIN Elite ABC Kit, Vector SG HRP Substrate (blue/gray).
Intestine substrate purple Intestine-substrate-purple Intestine-substrate-purple
Intestine: • Desmin (m), VECTASTAIN Elite ABC Kit, ImmPACT® DAB (brown) substrate. • Cytokeratin (m), VECTASTAIN Elite ABC Kit, Vector VIP (purple) substrate.
Tonsil substrate red Tonsil-substrate-red Tonsil-substrate-red
Tonsil: • Cyclin A (m), VECTASTAIN Universal ABC-AP Kit, Vector Blue AP Substrate (blue) • CD20 (m), VECTASTAIN Universal Elite ABC Kit, Vector NovaRED® HRP Substrate (red).
Breast Carcinoma substrate blue gray Breast_Carcinoma-substrate-blue-gray Breast_Carcinoma-substrate-blue-gray
Breast Carcinoma: • Estrogen Receptor (m); VECTASTAIN Elite ABC Kit, Vector NovaRED substrate (red) • CD34 (m), VECTASTAIN Elite ABC Kit, DAB substrate (brown) • Cytokeratin 8/18 (m), VECTASTAIN Elite ABC Kit, Vector SG substrate (blue/gray).
Tonsil substrate blue gray Tonsil-substrate-blue-gray Tonsil-substrate-blue-gray
Tonsil: • CD3 (m), VECTASTAIN Universal Elite ABC Kit, Vector VIP substrate (purple) • CD20 (m), VECTASTAIN®Universal Elite ABC Kit, Vector SG substrate (blue/gray).