Immunohistochemistry (IHC) Antibodies
Plan robust IHC on FFPE or frozen sections: choose targets, retrieval, and detection chemistries. This page curates commonly used markers across oncology, immune, neural, epithelial/EMT, endothelial, organ‑specific panels, and mismatch repair (MMR) with expected localization and typical antigen retrieval.
Key ideas: match fixation and antigen retrieval (HIER pH6 citrate vs pH9 Tris‑EDTA or enzymatic), block endogenous enzymes/biotin, pick HRP/DAB or AP/Fast Red chromogens, and interpret staining in the correct cellular compartment (nuclear, membranous, cytoplasmic).
Epithelial Core Markers
Pan‑cytokeratin plus CK7/CK20 patterns help define epithelial origin and guide tumor classification.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| Pan‑Cytokeratin (AE1/AE3) | Cytoplasmic | pH 6 | Broad epithelial screening marker | Pan‑CK antibodies |
| CK7 (KRT7) | Cytoplasmic | pH 6 | CK7+/CK20− pattern in many adenocarcinomas | CK7 antibodies |
| CK20 (KRT20) | Cytoplasmic | pH 6 | CK7−/CK20+ pattern in colorectal origin | CK20 antibodies |
| p63 (TP63) | Nuclear | pH 9 | Squamous differentiation; basal cells | p63 antibodies |
| p40 (ΔNp63) | Nuclear | pH 9 | More specific squamous marker than p63 | p40 antibodies |
Immune Markers
Combine pan‑leukocyte with T/B cell markers and macrophage markers to profile infiltrates; add PD‑L1 for checkpoint status.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| CD45 (PTPRC) | Membrane | pH 6 | Pan‑leukocyte marker | CD45 antibodies |
| CD3 (CD3E) | Membrane | pH 9 | T‑cell marker | CD3 antibodies |
| CD20 (MS4A1) | Membrane | pH 9 | B‑cell marker | CD20 antibodies |
| CD68 | Cytoplasmic/lysosomal | pH 6 | Macrophage marker | CD68 antibodies |
| CD163 | Membrane | pH 6 | M2 macrophage associated | CD163 antibodies |
| PD‑L1 (CD274) | Membrane | pH 9 | Checkpoint ligand; clone‑specific scoring varies | PD‑L1 antibodies |
| FOXP3 | Nuclear | pH 9 | Regulatory T‑cells | FOXP3 antibodies |
Proliferation & Apoptosis
Pair proliferation indices with tumor suppressor/oncogene status and apoptosis readouts.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| Ki‑67 (MKI67) | Nuclear | pH 9 | Scored as % positive nuclei | Ki‑67 antibodies |
| p53 (TP53) | Nuclear | pH 9 | Overexpression patterns correlate with mutation | p53 antibodies |
| Cleaved Caspase‑3 | Cytoplasmic/nuclear | pH 6 | Apoptosis marker (active fragments) | Caspase‑3 antibodies |
| BCL2 | Cytoplasmic/mitochondrial | pH 6 | Anti‑apoptotic; lymphoma panels | BCL2 antibodies |
Oncogenic Pathways
Common IHC targets in oncology diagnostics and research; interpret with appropriate scoring systems.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| HER2 (ERBB2) | Membrane | pH 9 | ASCO/CAP scoring (0–3+) | HER2 antibodies |
| EGFR | Membrane | pH 6/9 | Variable expression; tumor type dependent | EGFR antibodies |
| ER (ESR1) | Nuclear | pH 9 | Breast cancer; Allred score | ER antibodies |
| PR (PGR) | Nuclear | pH 9 | Breast cancer; complements ER | PR antibodies |
Endothelial & Angiogenesis
Vessel identification and angiogenic signaling markers.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| CD31 (PECAM1) | Membrane/junctions | pH 6 | Endothelial cell borders | CD31 antibodies |
| CD34 | Membrane | pH 6 | Endothelial & progenitor marker | CD34 antibodies |
| Von Willebrand Factor (VWF) | Cytoplasmic/granular | pH 6 | Weibel–Palade bodies | VWF antibodies |
| VEGF‑A (VEGFA) | Cytoplasmic/secreted | pH 6 | Angiogenic ligand | VEGFA antibodies |
Neural / Neuroendocrine
Core neural markers and classic neuroendocrine markers used in pathology.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| NeuN (RBFOX3) | Nuclear | pH 6 | Neuronal nuclei | NeuN antibodies |
| GFAP | Cytoplasmic | pH 6 | Astrocytes | GFAP antibodies |
| Iba1 (AIF1) | Cytoplasmic | pH 6 | Microglia/macrophages in CNS | Iba1 antibodies |
| Chromogranin A (CHGA) | Cytoplasmic/granular | pH 6 | Neuroendocrine secretory granules | CHGA antibodies |
| Synaptophysin (SYP) | Cytoplasmic/vesicular | pH 6 | Neuroendocrine & neuronal synapses | SYP antibodies |
Liver Panel
Markers helpful for hepatocellular differentiation and tumor origin.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| HepPar‑1 | Cytoplasmic | pH 6 | Hepatocellular marker | HepPar‑1 antibodies |
| Arginase‑1 (ARG1) | Cytoplasmic | pH 6 | Specific for hepatocytes | ARG1 antibodies |
| Glypican‑3 (GPC3) | Membrane/cytoplasmic | pH 6 | Hepatocellular carcinoma | GPC3 antibodies |
Lung Panel
Pulmonary adenocarcinoma vs squamous differentiation.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| TTF‑1 (NKX2‑1) | Nuclear | pH 9 | Lung adenocarcinoma marker | TTF‑1 antibodies |
| Napsin A (NAPSA) | Cytoplasmic/granular | pH 9 | Type II pneumocytes; adenocarcinoma | Napsin A antibodies |
| p40 (ΔNp63) | Nuclear | pH 9 | Squamous differentiation | p40 antibodies |
Renal Panel
Renal origin and differentiation markers.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| PAX8 | Nuclear | pH 9 | Renal/thyroid/Müllerian origin | PAX8 antibodies |
| WT1 | Nuclear | pH 9 | Renal development; mesothelial marker | WT1 antibodies |
| RCC marker (RCC Ma) | Membrane | pH 6 | Renal cell carcinoma antigen | RCC antibodies |
GI / Pancreas Panel
Intestinal differentiation and pancreatobiliary markers.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| CDX2 | Nuclear | pH 9 | Intestinal differentiation | CDX2 antibodies |
| MUC2 | Cytoplasmic | pH 6 | Goblet/mucinous tumors | MUC2 antibodies |
| MUC1 | Membrane | pH 6 | Pancreatobiliary/many adenocarcinomas | MUC1 antibodies |
Melanocytic Markers
Diagnostic panel for melanocytic differentiation; use a combination for sensitivity/specificity.
| Target (Gene) | Localization | Typical retrieval | Notes | Browse |
|---|---|---|---|---|
| S100 | Nuclear/cytoplasmic | pH 6 | Very sensitive; less specific | S100 antibodies |
| SOX10 | Nuclear | pH 9 | Specific for melanocytic/schwannian | SOX10 antibodies |
| Melan‑A (MLANA) | Cytoplasmic | pH 6 | Specific melanocytic marker | Melan‑A antibodies |
| HMB‑45 (PMEL) | Cytoplasmic | pH 6 | Melanosome‑associated; variable sensitivity | HMB‑45 antibodies |
Mismatch Repair (MSI) Panel
Loss of nuclear staining for any component (with positive internal control) suggests deficiency.
| Target (Gene) | Localization | Typical retrieval | Interpretation | Browse |
|---|---|---|---|---|
| MLH1 | Nuclear | pH 9 | Loss indicates MLH1 deficiency | MLH1 antibodies |
| MSH2 | Nuclear | pH 9 | Loss indicates MSH2 deficiency | MSH2 antibodies |
| MSH6 | Nuclear | pH 9 | Loss indicates MSH6 deficiency | MSH6 antibodies |
| PMS2 | Nuclear | pH 9 | Loss indicates PMS2 deficiency | PMS2 antibodies |
IHC Workflow Overview
A high‑level view of typical IHC steps; adapt times and concentrations to tissue type and antibody.
| Step | Typical options | Notes |
|---|---|---|
| Deparaffinize & rehydrate | Xylene → graded ethanol → water | For FFPE sections only. |
| Antigen retrieval | HIER pH6 citrate or pH9 Tris‑EDTA; enzymatic (Proteinase K, Trypsin) | Choose per epitope; see table below. |
| Block endogenous activity | Peroxidase (3% H2O2), alkaline phosphatase inhibitors; avidin/biotin block | Required for HRP/AP and biotin systems. |
| Blocking (protein/Fc) | Serum/BSA; Fc block for immune tissues | Reduces non‑specific binding. |
| Primary antibody | Optimized dilution; 30–60 min RT or overnight 4°C | Use validated clones/polyclonals for IHC. |
| Secondary / Polymer | HRP or AP polymers; biotin‑streptavidin | Polymers simplify and increase sensitivity. |
| Chromogen & counterstain | DAB (brown) / AEC or Fast Red (red); Hematoxylin | Pick contrast vs tissue pigment. |
| Mounting | Aqueous for AEC/Fast Red; resin for DAB | Chromogen chemistry dictates medium. |
Fixation & Antigen Retrieval
A quick guide to common retrieval conditions. Always start with manufacturer data and optimize locally.
| Target type | Typical retrieval | Specimen | Notes |
|---|---|---|---|
| Nuclear transcription factors | HIER pH 9 (Tris‑EDTA) | FFPE | Enhances nuclear epitope exposure (e.g., TTF‑1, ER). |
| Membrane receptors | HIER pH 6 (Citrate) or pH 9 | FFPE | Test both buffers (e.g., EGFR, HER2). |
| Cytoskeletal proteins | HIER pH 6 | FFPE / Frozen | Intermediate filaments and keratins. |
| Phospho‑epitopes | HIER pH 9; add phosphatase inhibitors | FFPE / Frozen | Minimize time in aqueous steps post‑fix. |
| Extracellular matrix | Enzymatic retrieval (Proteinase K/Trypsin) | FFPE | Harsh treatment—monitor morphology. |
| Frozen sections | No retrieval or mild HIER | Frozen | Fix with cold acetone or PFA as needed. |
Detection Systems & Chromogens
Choose HRP/DAB for permanence and contrast; AP/Red when melanin or heme obscures brown signal, or for dual‑stain contrast.
| System | Chromogen | Pros | Considerations |
|---|---|---|---|
| HRP polymer (anti‑mouse/rabbit) | DAB (brown) | High sensitivity; archival stability | Block endogenous peroxidase; resin mount |
| AP polymer | Fast Red / Permanent Red | Great in pigmented/hemorrhagic tissue | Aqueous mountants; lower photo‑stability |
| Biotin‑streptavidin | DAB, AEC, etc. | Flexible; legacy workflows | Block endogenous biotin (liver, kidney) |
Controls & Blocking
Use tissue‑appropriate positive controls and multiple negatives to interpret specificity clearly.
| Control | Purpose | Notes |
|---|---|---|
| Known positive tissue | Verifies assay detects target | Include retrieval/detection identical to test |
| No primary control | Non‑specific secondary/polymer | Should be blank after blocking steps |
| Isotype control | Background from primary isotype | Match species/isotype and concentration |
| Peroxidase/AP block | Reduce endogenous enzyme signal | 3% H2O2 (HRP); AP inhibitors as needed |
| Avidin/biotin block | Reduce endogenous biotin | Important in liver, kidney, brain |
Recommended Polymers / Secondary Systems
Species‑specific HRP/AP polymers simplify IHC and boost sensitivity vs traditional biotin systems.
| Primary host | Recommended system | Chromogen options | Notes |
|---|---|---|---|
| Rabbit | Anti‑Rabbit HRP/AP polymer | DAB, AEC/Fast Red | High affinity; minimal steps |
| Mouse | Anti‑Mouse HRP/AP polymer | DAB, AEC/Fast Red | For mouse tissue: consider mouse‑on‑mouse kits |
| Goat | Polymer via anti‑Goat bridge | DAB, AEC/Fast Red | Use donkey anti‑Goat bridges |
| Rat | Anti‑Rat HRP/AP or bridging | DAB, AEC/Fast Red | Cross‑adsorbed to reduce background |
IHC FAQs
Quick answers to common IHC questions.
| Question | Answer |
|---|---|
| Which retrieval buffer should I start with? | Begin with pH 6 citrate for most cytoplasmic/membrane proteins and pH 9 Tris‑EDTA for nuclear transcription factors. Optimize based on datasheet and tissue response. |
| When should I choose AP/Red over HRP/DAB? | Use AP/Red in pigmented tissues (melanin) or blood‑rich areas where brown signal may be obscured, or when performing dual‑color IHC for chromogen contrast. |
| How do I reduce background? | Increase protein/Fc blocking, ensure thorough peroxidase block, shorten primary incubation, increase wash times, and use species‑appropriate, cross‑adsorbed polymers. |
Build an IHC panel
Tell us your tissue, fixation (FFPE/frozen), retrieval system, and targets — we’ll recommend validated primaries and HRP/AP detection to match.