首页
400-620-6333
For IHC (Immunohistochemistry)
Immunohistochemistry (IHC) integrates tissue morphology with immunology to visualize target molecule expression on tissue sections via antigen–antibody specific recognition. It is widely used in basic research, drug development, pathological validation, and digital pathology analysis. IHC is highly sensitive to the chemical conditions of each step—sample pretreatment, antigen retrieval, blocking, antibody incubation, chromogenic/fluorescent detection, and mounting; any improper formulation, unstable impurity levels, or batch-to-batch fluctuation can lead to elevated background, signal shift, or localization distortion, thereby affecting reproducibility and cross-platform comparability.
I. Definition and Significance
“Reagents for IHC” refers to chemical and biological formulation systems validated for intended use across the full IHC workflow, covering key steps such as antigen retrieval of fixed sections, blocking, antibody dilution and incubation, washing, chromogenic/fluorescent detection, and mounting. Compared with general reagents, these products emphasize:
- Low background and high S/N: low-peroxide detergents, low-background chromogenic systems, and low-autofluorescence mounting media.
- Antigen fidelity and repeatability: precisely controlled retrieval buffer pH/capacity, high slide adhesion, and cross-batch consistency.
- System compatibility: clear do-nots for HRP/AP routes (HRP prohibits NaN₃; AP avoids phosphates throughout—incubation/washing/chromogenic).
- Complete documentation: COA including pH/purity/background and compatibility statements, with trend data and retention strategy.
II. Key Quality Requirements and Test Methods
Control Dimension | Quality Requirement | Test Method | Technical Significance |
Antigen epitope preservation | Compatible with heat- or enzyme-induced retrieval; stable antigen conformation | Standard positive tissue comparison; antigen recovery curves | Improve stability of positive signals |
Low background & non-specificity | Reduce endogenous enzymes, Fc, charged/hydrophobic adsorption | No-primary/isotype controls at equal concentration; background OD scan | Increase S/N and interpretation accuracy |
Enzyme/chromogenic compatibility | Smooth curves for HRP/DAB and AP/red substrates | Time-resolved chromogenic curves; inhibition tests | Maintain tone linearity and repeatability |
Fluorescence compatibility | Low autofluorescence; anti-fading | Autofluorescence baseline; photobleaching tolerance test | Fit for multiplex fluorescent IHC |
pH/buffer & salinity stability | Stable chemistry of diluents, washes, and blockers | pH, conductivity, and osmolality measurements | Prevent edge effects and artifacts |
Batch consistency | Functional release and trend monitoring | Negative/weak-positive/strong-positive three-level controls; inter-batch overlays | Ensure cross-batch comparability and retrospective consistency |
III. Scope of Application
- Routine localization and method validation: localization of membrane/cytoplasmic/nuclear markers in tissues/cells; small-scale optimization of retrieval (pH 6/9, time/temperature) and chromogenic to achieve maximal S/N.
- Tumor-related research: typing and origin determination (CK7/CK20, EMA, Syn/CgA), semi-quantitative evaluation of proliferation/pathway markers (Ki-67, p53, HER2, PD-L1) (for research use).
- Immune microenvironment assessment: infiltration and distribution of immune cells (CD3/CD4/CD8, CD20, CD68, FOXP3), with optional quantitative analysis via digital pathology.
- Organ and nervous system markers: localization and change observation of neural/glial (NeuN, GFAP, Iba1) and hepatic/renal/cardiopulmonary functional proteins (Albumin, Na⁺/K⁺-ATPase, AQP).
IV. Main Components and Functional Positioning
Reagent Category | Primary Function and Description | Common Examples |
The core reagent that specifically binds to the target antigen. | Monoclonal Antibody:High specificity, recognizes a single epitope. Polyclonal Antibodies:High affinity, recognizes multiple epitopes. | |
Binds specifically to the primary antibody; conjugated to a label for signal detection and amplification. | Enzyme-conjugated: e.g., HRP or AP labeled. Fluorophore-conjugated: e.g., Cy3, FITC labeled. | |
Detection System | Further amplifies the signal to enhance detection sensitivity, especially for low-abundance antigens. | Avidin-Biotin Complex (ABC) reagents. Polymer-based detection systems. |
Antigen Retrieval Reagent | Reverses formaldehyde-induced cross-linking to expose antigen epitopes. | Citrate buffer (pH 6.0). EDTA buffer(pH 8.0-9.0). |
Enzyme Substrate | Reacts with the conjugated enzyme (HRP/AP) to produce an insoluble colored precipitate for antigen localization. | |
Buffers & Blocking Solution | Used for washing, diluting, and blocking to reduce non-specific background staining. | Blocking Solution:e.g., normal serum, BSA/casein, endogenous peroxidase block (H₂O₂) |
Counterstain & Mounting Medium | Provides contrast staining for nuclei or cytoplasm and preserves the tissue section under a coverslip. | Counterstain:e.g., Hematoxylin. Mounting Medium: For fluorescence or permanent mounting. |
V. Common Experimental Problems and Solutions
Problem | Typical Manifestation | Possible Cause | Solution & Prevention |
High background | Diffuse light staining; non-target area coloration | Insufficient blocking; inadequate washing; mismatched diluent system | Strengthen blocking and washing; switch to low-background dilution/wash systems; optimize slide adhesion and cleanliness |
Weak/lost signal | Weak positives indistinct; blurred localization | Insufficient retrieval or antibody inactivation; aged sections | Prolong/adjust retrieval; verify antibody storage and in-use period; prefer matched retrieval buffers |
Over-retrieval | Elevated background; structural damage | Excessive heat retrieval; overlong enzyme retrieval | Lower temperature/time; switch to milder chemical retrieval |
Non-specific speckles/granules | Granular deposits; edge pseudo-positivity | Improper ionic strength in washing; slide contamination | Adjust ionic strength and surfactant level; use cleaner consumables |
VI. FAQs
Q1: Use pH 6 or pH 9 for retrieval?
A: Depends on the antibody and epitope. Generally, pH 6 (citrate) suits classic epitopes; pH 9 (EDTA) is more effective for membrane proteins/strong crosslinking.
Q2: Why is NaN₃ not allowed for the HRP route?
A: Sodium azide irreversibly inhibits HRP, directly causing signal reduction or false negatives. If a preservative is needed, select azide-free systems and verify by functional assays.
Q3: Why avoid phosphates during AP chromogenic stages?
A: Phosphate inhibits AP activity; use TBS throughout (incubation/washing/chromogenic) and confirm PO₄³⁻ is below the threshold in COA or spot checks.
VII. Aladdin Product Advantages
- Complete portfolio: primary/secondary antibodies, polymer HRP/AP detection, DAB and fluorescent substrates, retrieval/blocking/wash solutions, and controls—one-stop coverage of the IHC workflow.
- Low background, high S/N: formulations optimized for FFPE with parameter recommendations; validated on real tissues to significantly reduce non-specific staining.
- Stable and traceable batches: strict release and retained-sample comparison; COA provided with shipment/online, full batch traceability.
- Robust technical support: one-on-one condition optimization and multiplex IHC/IF design; guidance for migration to automated platforms.
VIII. Differences from Adjacent Grades
Grade | Core Application Scenario | Detection Target | Typical Sample Type | Core Advantage |
for IHC (Immunohistochemistry) | Protein localization and expression analysis at the tissue level | Spatial distribution of specific proteins in tissue cells | Formalin-fixed paraffin-embedded tissue, frozen tissue | Preserves tissue morphology; enables intuitive observation of protein localization in tissue |
for WB (Western Blot) | Protein quantification and molecular weight identification | Expression level and molecular weight confirmation of specific proteins | Cell lysates, tissue homogenates | Verifies protein molecular weight; suitable for semi-quantitative analysis |
Precise localization of proteins within cells/tissues | Subcellular localization of specific proteins | Cell climbing slides, frozen tissue, paraffin-embedded tissue | High resolution; enables simultaneous labeling of multiple proteins (multiplex fluorescence) | |
for ELISA (Enzyme-Linked Immunosorbent Assay) | Quantitative detection of proteins in body fluids | Absolute content of specific proteins (e.g., cytokines) | Serum, plasma, cell culture supernatant | High sensitivity, high throughput; suitable for large-scale sample quantification |
for IP (Immunoprecipitation) | Protein interaction studies and protein purification | Specific proteins and their interacting partners | Cell lysates | Captures target proteins and their complexes; facilitates protein interaction research |
In summary, For IHC reagents center on low background, high S/N, and cross-batch consistency, with intended-use validation around key steps such as antigen retrieval, blocking, incubation, chromogenic/fluorescence, and mounting, ensuring credible localization, reproducible results, and platform comparability. Aladdin provides systematized products and methodological support across the full IHC workflow, and through rigorous QC and traceable documentation—covering both manual and automated scenarios—helps users obtain stable and interpretable staining results more efficiently.
Aladdin: https://www.aladdinsci.com/