Enzyme Substrate Combinations

Immunohistochemistry Resource Guide

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Enzyme Substrate Combinations

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

Substrate Combinationscrop
Key: Alkaline Phosphatase Peroxidase + Indicates good contrast – Indicates incompatiblity of substrates for various reasons
Breast Carcinoma Brown
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 Blue Gray
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 Red
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 Red
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 Sg Hrp Substrate Gray
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 Vector Vip Purple
Breast Carcinoma: • Ki67 (rm), ImmPRESS Reagent (HRP; Universal), Vector DAB (brown) • CD34 (m), ImmPRESS Reagent (HRP; Universal), Vector VIP (purple).
Melanoma Brown Pigments
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 Vector Vip Purple
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 Hrp Substrate Gray Black
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 Vector Blue Ap Substrate Blue
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 Sg Hrp Substrate Blue Gray
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).

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