
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
Enzyme Substrate Combinations
Enzyme Substrate Combinations
Recommended enzyme substrate combinations and the recommended order in which they should be used.

Second Substrate |
ImmPACT™ Vector® Red & Vector® Red (magenta) SK-5105 & SK-5100 | Vector® Blue (blue) SK-5300 | BCIP/NBT (indigo) SK-5400 | ImmPACT™ VIP & Vector® VIP (purple) SK-4605 & SK-4600 | ImmPACT™ DAB, mmPACT™ DAB EqV & Vector® DAB (brown) SK-4105, SK-4103, SK-4100 | DAB-Ni (gray-black) SK-4100 | ImmPACT™ NovaRED™ & Vector® NovaRED™ (red) SK-4805 & SK-4800 | ImmPACT™ SG & ector® SG (blue-gray) SK-4705 & SK-4700 | ImmPACT™ AEC & Vector® AEC (red) SK-4205 & SK-4200 | ImmPACT™ AMEC Red (red) SK-4285 |
---|---|---|---|---|---|---|---|---|---|---|
First Substrate |
||||||||||
ImmPACT™ Vector® Red & Vector® Red (magenta) Cat. No. SK-5105 & SK-5100 | – | – | – | + | + | – | + | – | – | |
Vector® Blue (blue) Cat. No. SK-5300 | + | – | + | + | + | + | + | + | + | |
BCIP/NBT (indigo) Cat. No. SK-5400 | + | – | + | + | + | + | + | + | + | |
ImmPACT™ VIP & Vector® VIP (purple) Cat. No. SK-4605, SK-4600 | – | + | – | + | + | – | + | – | – | |
ImmPACT™ DAB, ImmPACT™ DAB EqV & Vector® DAB (brown) Cat. No. SK-4105, SK-4103, SK-4100 | + | + | + | + | – | – | + | + | + | |
Vector® DAB-Ni (gray-black) Cat. No. SK-4100 | + | – | – | + | + | + | – | – | – | |
ImmPACT™ NovaRED™ & Vector® NovaRED™ (red) Cat. No. SK-4805, SK-4800 | – | + | + | – | + | + | + | – | – | |
ImmPACT™ SG & Vector® SG (blue-gray) Cat. No. SK-4705, SK-4700 | + | – | – | + | + | – | – | + | + | |
ImmPACT™ AEC & Vector® AEC (red) Cat. No. SK-4205, SK-4200 | – | – | – | – | + | – | – | + | – | |
ImmPACT™ AMEC Red (red) SK-4285 | – | – | – | – | + | – | – | + | – |
Key: Alkaline Phosphatase Peroxidase + Indicates good contrast – Indicates incompatiblity of substrates for various reasons
.

Breast Carcinoma: • Estrogen Receptor (m), VECTASTAIN® Elite® ABC Kit, Vector® NovaRED® HRP substrate (red) • CD34 (m), VECTASTAIN ABC-AP Kit, Vector Blue AP Substrate (blue) • Cytokeratin 8/18 (m), VECTASTAIN Elite ABC Kit, Vector DAB HRP Substrate (brown).

Tonsil: • CD3 (m), ImmPRESS® Anti-Mouse IgG Reagent, Vector VIP HRP Substrate (purple) • Ki67 (m), ImmPRESS Anti-Mouse IgG Reagent, Vector SG HRP Substrate (blue/gray).

Prostate: • Cytokeratin 5 (m), VECTASTAIN Universal ABC-AP Kit, Vector Blue AP Substrate (blue) • CD34 (m), VECTASTAIN Universal ABC-AP Kit, Vector Red AP Substrate (red).
Multiple Labeling Examples

Colon Cancer: • Ki67 (rm), ImmPRESS-AP Anti-Rabbit IgG Reagent, Vector Blue AP Substrate (blue) • Cox2 (rm), ImmPRESS-AP Anti-Rabbit IgG Reagent, ImmPACT® Vector Red AP Substrate (red).

Colon: • Ki67 (rm), ImmPRESS Universal (Anti-Mouse/Anti-Rabbit IgG) HRP Reagent, Vector VIP (purple) • Cytokeratin (m), ImmPRESS Universal (Anti- Mouse/Anti-Rabbit IgG) HRP Reagent, Vector SG HRP Substrate (gray).

Breast Carcinoma: • Ki67 (rm), ImmPRESS Reagent (HRP; Universal), Vector DAB (brown) • CD34 (m), ImmPRESS Reagent (HRP; Universal), Vector VIP (purple).

Melanoma: • Cyclin A (m), ImmPRESS-AP Anti-Mouse IgG, ImmPACT Vector Red AP Substrate (magenta) • Melanoma Marker (m) ImmPRESS HRP Anti- Mouse IgG, Vector SG HRP Substrate (gray). Note contrast of double stain with the brown pigments in the tissue.

Tonsil: • CD3 (m), VECTASTAIN Elite ABC Kit (Universal), Vector DAB (brown) • CD20 (m), VECTASTAIN Elite ABC Kit (Universal), Vector SG (blue-gray) • Multi-Cytokeratin (m), VECTASTAIN Elite ABC Kit (Universal), Vector VIP (purple).

Breast Carcinoma: • Estrogen Receptor (rm), ImmPRESS Universal Reagent, Vector NovaRED HRP Substrate (red) • M2A Antigen (m), ImmPRESS Universal HRP Reagent, Vector DAB+Ni HRP Substrate (gray/black).

Colon: • M2A Antigen (m), ImmPRESS Universal (Anti-Mouse/Rabbit IgG) HRP Reagent, Vector NovaRED HRP Substrate (red) • CD34 (m), VECTASTAIN Universal ABC-AP kit, Vector Blue AP Substrate (blue).

Colon Carcinoma: • S100 (rp), VECTASTAIN Universal ABC-AP Kit, Vector Red AP Substrate (red) • Cytokeratin 8/18 (m), VECTASTAIN Universal Elite ABC Kit, Vector SG HRP Substrate (blue/gray).