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Enzyme Substrates

A Palette of Colors for Visualizing Target Antigens

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Achieve Crisp, Precise, and Sensitive Staining with High-Performance Chromogenic Substrates

Chromogenic enzyme substrates play a crucial role in immunodetection applications, allowing researchers to visualize target antigens with precise colorimetric staining. Chromogenic substrates generate visible color upon reaction with enzyme detection reagents, enabling precise localization of target antigens. This makes enzyme substrates fundamental to immunohistochemistry (IHC) and related techniques, such as blotting applications, ELISA, ELISpot, and in situ hybridization (ISH).

Vector Laboratories is an industry leader in developing and manufacturing high-performing chromogenic substrates that provide superior sensitivity and reliability. Vector offers high-performing versions of substrates like DAB, while also expanding the landscape of chromogenic detection with a portfolio of novel, proprietary substrates. All Vector enzyme substrates are designed to meet the needs of diverse research applications and are optimized for sensitivity, signal clarity, reproducibility, and ease of use.

Comparison of Enzyme Substrate Characteristics

Vector Laboratories provides a wide range of enzyme substrates with diverse performance characteristics, allowing researchers to select the best substrate for their applications. With options that span chemistry, microscopy techniques, mounting media compatibility, multiplex capability, and heat resistance, there is a Vector enzyme substrate for every experimental design. The table below provides the characteristics of our enzyme substrate offerings to help you determine the optimal substrate for your application.

Substrate

Color

Catalog Number

Microscopy

Mounting

Contrast in Pigmented Tissue

Multiple Labeling

Heat Resistant*

Brightfield

Darkfield

Electron

Fluo-
rescence

Spectral Imaging

Peroxidase

DAB

table brown

Brown

SK-4100

table ok

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

DAB+Ni

table grey black

Gray-Black

SK-4100

table ok

table ok

table ok

table ok

Non-aqueous

table ok

ImmPACT DAB

table brown

Brown

SK-4105

table ok

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

ImmPACT DAB EqV

table brown

Brown

SK-4103

table ok

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

Vector VIP

table purple

Purple

SK-4600

table ok

table ok

table ok

table ok

Non-aqueous

table ok

table ok

ImmPACT VIP

table purple

Purple

SK-4605

table ok

table ok

table ok

table ok

Non-aqueous

table ok

table ok

Vector SG

table blue grey

Blue-Gray

SK-4700

table ok

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

ImmPACT SG

table blue grey

Blue-Gray

SK-4705

table ok

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

Vector NovaRED

table dark red

Red

SK-4800

table ok

table ok

table ok

table ok

Non-aqueous

table ok

table ok

ImmPACT NovaRED

table dark red

Red

SK-4805

table ok

table ok

table ok

table ok

Non-aqueous

table ok

table ok

AEC

table light red

Red

SK-4200

table ok

table ok

Aqueous

table ok

table ok

ImmPACT AEC

table light red

Red

SK-4205

table ok

table ok

Aqueous

table ok

table ok

ImmPACT AMEC Red

table light red

Red

SK-4285

table ok

table ok

Aqueous

table ok

table ok

TMB

table light blue

Blue

SK-4400

table ok

table ok

Non-aqueous

Alkaline Phosphatase

Vector Red

table magenta

Magenta

SK-5100

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

table ok

ImmPACT Vector Red

table magenta

Magenta

SK-5105

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

table ok

Vector Blue

table blue

Blue

SK-5300

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

table ok

Vector Black

table brown

Brown-Black

SK-5200

table ok

Non-aqueous

BCIP / NBT

table indigo

Indigo

SK-5400

table ok

table ok

table ok

Non-aqueous
or Aqueous

table ok

table ok

Are you selecting the best substrate for your Assay?

Vector’s Substrates Provide Solutions for Workflow Requirements

Selecting the appropriate enzyme substrate is crucial for successful immunodetection. Key factors to consider include:

FactorImportanceHow Vector Laboratories Delivers
Enzyme Compatibility
To create a chromogenic reaction, the substrate must match the detection enzyme (HRP or AP).Vector offers a full portfolio of HRP and AP substrates for flexibility in detection.
SensitivityHigher sensitivity allows detection of low-abundance targets and often enables dilution of primary antibody reagents.ImmPACT® substrates provide maximum sensitivity, reducing primary antibody consumption and cost per test.
Color ContrastHigh contrast enhances antigen visibility and prevents misinterpretation.Vector provides a wide range of colors to optimize contrast for various sample types.
Precipitate AppearanceThe appearance of precipitates in different substrates impacts clarity and localization of staining.HRP substrates form sharp, dense precipitates; AP substrates provide diffuse, translucent staining for broader expression analysis.
Workflow StabilityStable working solutions minimize variability and improve automation compatibility.Most ImmPACT® substrates’ working solutions remain stable for 14 days ensuring convenience and consistent performance in automated workflows.
Ease of UseFewer preparation steps simplify workflows and prevent potential variability.All Vector substrates come in ready-to-use liquid formats, eliminating the need for powders or tablets.
Multiplexing CapabilityMultiplexing allows for labeling of multiple antigens with distinct colors.Vector’s broad color options support multiplex IHC for clear differentiation of targets.
Heat ResistanceWorkflows requiring post-staining heating, such as ISH, must employ heat resistant reagents.Several Vector substrates are heat-resistant making them ideal for workflows requiring a heating step.
Mounting Media CompatibilityMounting media application preserves precipitate intensity, color, and clarity over time.Vector offers substrates compatible with both aqueous and non-aqueous mounting media.
Visualization MethodsCompatibility across different microscopy techniques provides experimental flexibility.Vector’s substrates are compatible with many different visualization methods.

Not all chromogenic substrates have equivalent sensitivity

While chromogenic detection methods follow the same basic principle, not every enzyme substrate offers the same sensitivity. For targets with low abundance, higher sensitivity enzyme substrates enhance signal detection. The table below shows the relative sensitivities of Vector enzyme substrates.

Chromogenic Substrate Sensitivity

enzyme substrates sensitivity table 2048x1269 1

* AEC & AMEC substrates must be aqueously mounted ** Longer incubation times increase sensitivity

Vector enzyme substrates demonstrate a range of sensitivities. This table compares the sensitivity of Vector enzyme substrates, including DAB substrates with enhanced sensitivity over competitor DAB tablets.

Not all DAB substrates are created equal

Vector’s ImmPACT® DAB provides superior sensitivity in side-by-side comparisons with competitor products. Greater signal intensity enables the visualization of low abundance targets and can often lead to a reduction in primary antibody concentrations, lowering the cost per test.

DAB (HRP) Substrate Sensitivity Comparison

Human tonsil (FFPE serial sections): Stained for CD20 (1:300 dilution), detected with ImmPRESS-HRP Anti-Mouse IgG (MP-7402). The only reagent variable was the DAB substrate used for color development.

ImmPACT DAB
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Company B
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Company D
es substrate sensitivity comparisons pic3 1024x695 1
Company E
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Company P
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Company S
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Overall Results: In parallel assays on serial sections from the same tissue block with only the DAB substrate being substituted, Vector ImmPACT DAB appeared to generate the most robust, crisp staining (highest sensitivity) without appreciable background compared with DAB from the other sources.

ImmPACT DAB
Primary antibody dilution:   1:50
es substrate sensitivity comparisons pic7
1:100
es substrate sensitivity comparisons pic8
1:400
es substrate sensitivity comparisons pic9
Company S
Primary antibody dilution:   1:50
es substrate sensitivity comparisons pic10
1:100
es substrate sensitivity comparisons pic11
1:400
es substrate sensitivity comparisons pic12

Comparison of IHC signal using Vector’s ImmPACT DAB and DAB from competitor Company S at various antibody concentrations. ImmPACT DAB demonstrates greater sensitivity and more intense signal at every concentration when compared to the other DAB.

Why Choose Vector Laboratories for Chromogenic Substrates?

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Scalable Manufacturing

Ensures batch-to-batch consistency for reliable reagent performance.

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Robust QC Testing

Each batch undergoes rigorous quality control for reproducible results.

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Proven Stability

Long shelf life and stable performance reduce experimental variability. 

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Industry Benchmarked

Recognized as a gold standard in IHC workflows, offering superior results. 

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ISO 9001-Certified

Demonstrates our commitment to high-quality standards and customer satisfaction.

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Expert Technical Support

Our knowledgeable team provides assay optimization, troubleshooting, and guidance at every step.

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Cited in High-Impact Journals

Widely referenced in scientific literature, reinforcing credibility and reliability.

Enzyme Substrate Technical and Educational Resources

To learn more about chromogenic enzyme substrates for immunodetection applications, explore the following resources.

IHC Multiplexing Guide

This comprehensive resource provides illustrated protocols, guidance on substrate pairing with photos, and practical tips for IHC multiplexing.

Download Guide

Enzyme Substrate Brochure

This brochure includes photomicrographs of substrate colors, a substrate properties table, a sensitivity schematic, and a compatibility guide for multiplexing.

Learn More

Automated IHC Protocols using ImmPRESS Polymer Reagents

Review methodology for implementing ImmPRESS polymer reagents in a variety of automated staining platforms.

Download White Paper

Automated Staining Protocol with VectaPlex Antibody Removal Kit

Learn how VectaPlex and ImmPRESS enable multiplex staining on the Leica BOND automated stainer.

Download White Paper

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