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PDCD1LG2 / Programmed cell death 1 ligand 2 · IHC design guide
Design Immunohistochemistry for PDCD1LG2
Plan PDCD1LG2 IHC-P around variable cytoplasmic tissue staining (HPA tissue IHC). Start with the human IHC-validated antibody at 2 μg/mL (datasheet M03295), and account for a secreted variant when interpreting staining (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
PDCD1LG2 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Observed cytoplasmic (HPA tissue IHC); membrane and secreted forms annotated (UniProt) | |
| Staining pattern | Variable cytoplasmic staining across tissue cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet A03295) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Adipose tissue |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep formaldehyde fixation consistent (datasheet A03295 IHC) (selected-SKU IHC image A03295) | |
| Caveat | A secreted variant may weaken RNA–protein agreement (HPA tissue IHC) | |
| Regulation | Staining levels vary by tissue (HPA tissue IHC) | |
| Isoform / epitope | 3 isoforms; check extracellular versus cytoplasmic epitope coverage (UniProt) |
Section 1
Recommended PDCD1LG2 IHC & IF Protocols
The catalog antibody uses citrate pH 6 retrieval (datasheet A03295). The published protocols below describe PDCD1LG2 IHC in colorectal cancer tissue (PMC5668177; PMC6215508).
| Sample | Formaldehyde-fixed, paraffin-embedded mouse brain tissue (datasheet A03295) |
| Fixation | Image formalin-fixed; duration unreported (datasheet A03295); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6 (datasheet A03295); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% serum (datasheet A03295) |
| Primary antibody | Rabbit anti-PDCD1LG2, 2.5 μg/mL (datasheet A03295) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | PDCD1LG2-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression at variable levels in all tissues. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6 HIER for the catalog antibody (datasheet A03295); evaluate pH 8 retrieval when adapting the published protocol (PMC5668177).
Section 2
What Is the Expected PDCD1LG2 Staining Pattern?
In paraffin-section IHC, expect mainly cytoplasmic staining in tissue cells, with high staining reported in bronchial respiratory epithelium and several glandular or squamous epithelia (HPA: tissue IHC). A cell-surface component is biologically plausible because PDCD1LG2 has an extracellular domain and a transmembrane segment (UniProt Q9BQ51 topology). Treat the pattern cautiously: HPA rates the IHC antibody Approved but reports low consistency between staining and RNA expression (HPA: tissue IHC; HPA: HPA013411 IHC Approved).
| Strong cytoplasmic staining in bronchial respiratory epithelial cells, with nearby cells staining differently (HPA: High in bronchus respiratory epithelial cells). | This matches a documented positive pattern. Evaluate staining within the named cell population and tissue architecture; do not require every cell to match its intensity (HPA: variable cytoplasmic expression across tissues). |
| Predominantly nuclear staining, with little cytoplasmic signal in an expected positive cell population (HPA: tissue IHC cytoplasmic profile). | The compartment differs from the reported IHC pattern and UniProt membrane topology. Investigate nonspecific staining or image interpretation before calling PDCD1LG2 positive (HPA: tissue IHC; UniProt Q9BQ51 topology; general IHC practice). |
| A different cell type stains more strongly than the HPA-listed positive cells in the same section (HPA: tissue IHC positive-cell listings). | Check cell identity and controls. Cross-reactivity or endogenous detection activity can produce a misleading signal; intensity alone cannot assign it to PDCD1LG2 (general IHC practice). |
| Brown signal covers tissue, empty areas, or multiple cell types without a readable cellular pattern (general chromogenic IHC practice). | Diffuse background prevents a confident localisation call. Review the negative control, blocking, antibody concentration and wash conditions before comparing tissues (general IHC practice). |
| No signal in bronchial respiratory epithelial cells despite usable tissue morphology (HPA: High in bronchus respiratory epithelial cells). | This is a failed expected-positive check, not proof of absent PDCD1LG2. Review assay controls and detection conditions; HPA reports variable tissue staining and low RNA–protein consistency (HPA: tissue IHC; general IHC practice). |
💡Expected PDCD1LG2 appearanceCall a convincing positive result when identifiable HPA-listed cells show cytoplasmic staining, potentially high in bronchial respiratory epithelium; isolated nuclear colour or diffuse colour without cell boundaries is suspect (HPA: tissue IHC; general IHC practice).
| Cell compartment and epitope context (UniProt Q9BQ51 topology). | The mature chain spans residues 20–273, with extracellular residues 20–220 and a transmembrane segment at 221–241. Epitope position could affect which protein pool an antibody detects; the supplied record gives no antibody epitope (UniProt Q9BQ51 topology). |
| Secreted protein variant (HPA: tissue IHC reliability note). | HPA notes that at least one variant is secreted, so protein location can differ from RNA location. Interpret cell-level staining alongside morphology; RNA enrichment alone does not establish an IHC-positive compartment (HPA: tissue IHC reliability note). |
| Isoforms and processing (UniProt Q9BQ51). | UniProt lists three isoforms and a signal peptide at residues 1–19. The supplied evidence does not assign the observed IHC pattern to a particular isoform or show which forms the antibody detects (UniProt Q9BQ51 isoforms and processing). |
| Glycosylation (UniProt Q9BQ51 PTMs). | Five glycosylation sites are annotated at residues 37, 64, 157, 163 and 189. Their presence does not establish a particular retrieval setting or fixation sensitivity for this antibody (UniProt Q9BQ51 glycosylation; evidence scope). |
| Tissue and antibody evidence (HPA: tissue IHC; HPA: HPA013411 validation). | The tissue profile reports variable cytoplasmic staining, while HPA013411 is Approved for IHC. HPA also reports low staining–RNA consistency; use listed cell types as checks, not an expression ranking inferred from RNA (HPA: tissue IHC). |
| IF/ICC Q: Does cytosolic IF confirm the paraffin IHC result? (HPA: subcellular; HPA: HPA013411 validation). | A: HPA calls the cytosol location uncertain and HPA013411 ICC validation Uncertain. Treat IF/ICC as separate evidence; its U-251MG and U2OS images do not validate an IHC retrieval or detection choice (HPA: subcellular; HPA: HPA013411 validation). |
| Situation | Likely cause | Next action |
|---|---|---|
| Bronchial epithelium is blank, while the run's other controls develop colour (HPA: High in bronchus respiratory epithelial cells). | The expected-positive check has failed; the supplied sources do not identify a PDCD1LG2-specific retrieval or fixation failure mechanism (HPA: tissue IHC; evidence scope). | Verify tissue identity, antibody handling and the run control; review retrieval and detection as general IHC variables, then repeat with the documented positive tissue (general IHC practice; HPA: High in bronchus respiratory epithelial cells). |
| Nuclei dominate the signal in an otherwise interpretable section (HPA: tissue IHC cytoplasmic profile). | Nuclear dominance conflicts with the reported tissue profile; nonspecific detection or a reading error is possible (HPA: tissue IHC; general IHC practice). | Compare the negative control and counterstain, then reassess the cell boundaries before scoring any PDCD1LG2-positive cells (general IHC practice). |
| Adipocytes stain strongly (HPA: Not detected in adipose tissue adipocytes). | This conflicts with the listed negative cell population, although one negative listing cannot prove that every specimen must be blank (HPA: tissue IHC). | Check morphology, negative controls and background distribution; withhold a specific positive call until the unexpected signal is resolved (general IHC practice). |
| The whole section has widespread colour with little cellular contrast (general chromogenic IHC practice). | Background from detection reagents, insufficient blocking or inadequate washing may obscure a cell-specific pattern (general IHC practice). | Inspect reagent-only and negative controls; optimise blocking, washes and antibody concentration using the assay's existing instructions (general IHC practice). |
| IHC seems cytoplasmic, but an IF image appears different (HPA: tissue IHC; HPA: subcellular). | The assays have different evidence grades: HPA tissue IHC is Approved, while the cytosol IF location and HPA013411 ICC validation are Uncertain (HPA: tissue IHC; HPA: subcellular; HPA: HPA013411 validation). | Interpret each assay against its own controls and morphology; do not use uncertain ICC localisation to override the documented IHC tissue pattern (HPA: tissue IHC; HPA: subcellular; general IHC practice). |
| A tissue with high RNA expression gives weaker IHC staining than anticipated (HPA: tissue IHC reliability note). | HPA reports low agreement between antibody staining and RNA expression and notes a secreted variant; RNA level is an unreliable stain-intensity prediction here (HPA: tissue IHC reliability note). | Score the observed cells and compartment against HPA's tissue IHC profile, while recording discordance rather than changing a staining call to match RNA (HPA: tissue IHC; general IHC practice). |
Experiment evidence
IHC & IF images showing PDCD1LG2 staining
A03295 · IHCImmunohistochemistry Validation of PD-L2 in Mouse Brain Tissue Immunohistochemical analysis of paraffin-embedded mouse brain tissue using an
A03295 · IHCImmunohistochemistry Validation of PD-L2 in Mouse Brain Tissue Immunohistochemical analysis of paraffin-embedded mouse brain tissue using an
A03295 · IFImmunofluorescence Validation of PD-L2 in Mouse Brain Tissue Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain cells label
M03295-1 · IHCImmunohistochemical analysis of PDL-2 in untransfected(left) or transfected(right) with 293T cell sections. Cell was fixed with formaldehyde
M03295 · IHCImmunohistochemistry of PD-L2 in human tonsil tissue with PD-L2 antibody at 2 μg/mL.
M03295 · IHCImmunohistochemistry of PD-L2 in human colon carcinoma tissue with PD-L2 antibody at 2 μg/mL.
M03295 · IFImmunocytochemistry of PD-L2 in transfected HEK293 cells with PD-L2 antibody at 5 μg/mL. Lower left: Immunocytochemistry in transfected HEK2
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000197646-PDCD1LG2/tissue/cerebral+cortex#imid_3475456
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000197646-PDCD1LG2/tissue/colon#imid_3475382
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000197646-PDCD1LG2/tissue/liver#imid_3475375
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000197646-PDCD1LG2/tissue/pancreas#imid_3475326
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA013411; green = PDCD1LG2, blue = nucleus, red = microtubules.
HPA · ICC-IF U-251MGHuman Protein Atlas immunofluorescence, U-251MG, antibody HPA013411; green = PDCD1LG2, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for PDCD1LG2 IHC & IF
🧪Run appendix first and assess staining in glandular cells (HPA: High in appendix glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the appendix slide, neighboring cells chosen as internal negatives should show only control-level signal (standard IHC practice).
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PDCD1LG2 in U-251MG, U2OS, with annotated localisation: Cytosol (uncertain) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and host- and clonality-matched isotype controls, plus knockout tissue or a validated peptide-block control (standard IHC practice). For chromogenic detection in appendix, quench endogenous peroxidase and check for endogenous biotin if using avidin–biotin detection (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A03295 tissue-IHC record does not establish a fixative (selected-SKU fixative field: not stated). The paraffin-section example uses heat retrieval in citrate buffer at pH 6, but retrieval dependency is unreported (selected-SKU caption: citrate heat retrieval; standard IHC practice). Frozen sections or IF cannot be judged easier from the supplied evidence; in appendix, verify that glandular luminal material is not mistaken for cellular chromogen (standard IHC practice).
HPA tissue IHC evidence for PDCD1LG2
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced PDCD1LG2 IHC Tips
Troubleshoot PDCD1LG2 staining by checking retrieval, tissue morphology, staining compartment and cell-level controls (datasheet A03295; HPA; UniProt Q9BQ51).
What should I change when PDCD1LG2 staining is weak after antigen retrieval?
Start with heat-mediated citrate buffer at pH 6 for paraffin-section IHC (datasheet A03295). The selected tissue-IHC image used that retrieval before an overnight primary incubation at 4 °C, so reproduce those documented conditions when assessing a weak result (datasheet A03295). Record heating time and temperature, cooling time, section thickness and reagent age, then change one variable at a time (standard IHC practice). Compare the test section with a positive-control section processed in the same run, and check that morphology remains intact (standard IHC practice). If staining remains weak, test another retrieval condition as a fallback and assess signal alongside tissue damage and background (standard IHC practice).
How can fixation affect a weak or uneven PDCD1LG2 IHC result?
Target-specific sensitivity of PDCD1LG2 to fixation is unknown from the supplied evidence, so treat fixation changes as controlled IHC experiments rather than established remedies. Keep fixative, fixation interval, tissue processing and section thickness consistent across samples being compared (standard IHC practice). If staining differs between batches, review processing records and stain a known positive-control section in each run (standard IHC practice). Assess morphology and antibody-independent background before attributing a weak result to antigen loss (standard IHC practice). Do not infer a preferred fixation interval from the reported tissue staining pattern or from PDCD1LG2 membrane topology (HPA tissue IHC; UniProt Q9BQ51 topology).
Should PDCD1LG2 staining appear on the membrane or in the cytoplasm?
Assess membrane and intracellular staining separately: PDCD1LG2 has an extracellular region at residues 20–220 and a transmembrane segment at 221–241 (UniProt Q9BQ51 topology). UniProt also lists secreted and endomembrane locations, while tissue IHC reports variable cytoplasmic staining and subcellular imaging calls the cytosol assignment uncertain (UniProt Q9BQ51 subcellular; HPA tissue IHC; HPA subcellular). Therefore, record the compartment, cell type and staining intensity rather than treating every brown cytoplasmic deposit as equivalent to cell-surface expression (standard IHC interpretation). Compare each pattern with a positive-control section and a negative reagent control processed together (standard IHC practice). Interpret an isolated, unexpected compartment cautiously until an independent antibody or orthogonal assay supports it (standard IHC practice).
Could isoforms or epitope accessibility explain discordant PDCD1LG2 staining?
PDCD1LG2 has 3 annotated isoforms, but the supplied evidence does not identify the catalog antibody’s epitope or its recognition of each isoform (UniProt Q9BQ51 isoforms; datasheet A03295). Its extracellular region contains Ig-like domains, and glycosylation is annotated at residues 37, 64, 157, 163 and 189 (UniProt Q9BQ51 domains and glycosylation). Those features make epitope accessibility a question to test; they do not establish the cause of a particular IHC result (UniProt Q9BQ51; standard IHC interpretation). Check the antibody’s documented immunogen or epitope before assigning staining to an isoform, and compare independent antibodies where available (standard IHC practice). Keep retrieval and detection conditions matched when comparing their patterns (standard IHC practice).
How should I investigate a PDCD1LG2 pattern with multiplex IF?
Use IF as a separate validation experiment and include a marker for the cell type being assessed, such as an epithelial marker when examining bronchial respiratory epithelial cells (HPA: High in bronchial respiratory epithelial cells; standard IF practice). Choose spectrally separated fluorophores and consider a far-red channel for the weaker signal where tissue autofluorescence interferes (standard IF practice). PDCD1LG2 has an extracellular region at residues 20–220 and a cytoplasmic tail at 242–273 (UniProt Q9BQ51 topology). Plan permeabilisation around the antibody’s documented epitope: an accessible extracellular epitope may permit surface staining without it, whereas an intracellular epitope requires membrane access (standard IF practice). Include single-stain and secondary-only controls to evaluate bleed-through and background (standard IF practice).
What should I check when chromogenic PDCD1LG2 staining looks diffuse?
Separate tissue background from antibody-associated staining with a no-primary control and a positive-control section in the same run (standard IHC practice). The selected tissue-IHC image used 10% serum blocking for 1 hour at room temperature and an HRP secondary at 1:250; these are documented image conditions, not evidence that they eliminate background in every tissue (datasheet A03295). For an HRP and DAB workflow, check the peroxidase block and avoid excessive chromogen development (standard IHC practice). Inspect section edges, folds and necrotic areas before scoring diffuse color (standard IHC practice). If background persists, titrate the primary antibody while keeping retrieval and detection conditions fixed (standard IHC practice).
How should I quantify PDCD1LG2 staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, and apply the same rule to every section (standard IHC practice). For cellular staining, report the percentage of positive cells and an intensity-based H-score; for sparse positive cells, report positive-cell density per mm² of evaluable tissue (standard IHC quantification). Normalise counts to the relevant cell population or analyzable tissue area, excluding folds, necrosis and missing tissue by a prespecified rule (standard IHC practice). Keep retrieval, detection, counterstaining and image thresholds consistent across comparison groups (standard IHC practice). Report membrane and cytoplasmic scores separately because both membrane association and variable cytoplasmic tissue staining are documented (UniProt Q9BQ51 subcellular; HPA tissue IHC).
How can I distinguish credible PDCD1LG2 staining from artefact?
First identify the stained cell and compartment: high staining is reported in colon glandular cells, while the protein has membrane, endomembrane and secreted annotations (HPA: High in colon glandular cells; UniProt Q9BQ51 subcellular). A cytoplasmic pattern alone is not decisive because variable cytoplasmic tissue staining is reported and the cytosol assignment in subcellular imaging is uncertain (HPA tissue IHC; HPA subcellular). Check whether staining concentrates at section edges, folds or necrosis, and whether the no-primary control also develops color (standard IHC practice). Evaluate endogenous peroxidase background in the HRP workflow before calling diffuse DAB deposition positive (standard IHC practice). Seek reproducible cell-level staining across sections or support from an independent antibody before assigning an unexpected pattern to PDCD1LG2 (standard IHC practice).
Boster reagents
Best PDCD1LG2 / Programmed cell death 1 ligand 2 IHC Antibodies
Validated anti-PDCD1LG2 IHC images cover mouse brain (A03295 caption), human tonsil and colon carcinoma (M03295 captions), and 293T cell sections (M03295-1 caption); IF images cover mouse brain and human tissue (catalog IF captions).
Real IHC data
Anti-PD-L2 PDCD1LG2 Antibody
Cat # A03295
Real IHC data
Anti-PDCD1LG2 Antibody (N-term)
Cat # M03295-1
Real IHC data
Anti-PDL2 PDCD1LG2 Monoclonal Antibody [4E10]
Cat # M03295
A03295 shows IHC in formaldehyde-fixed paraffin-embedded mouse brain and IF in fixed mouse brain cells or tissue (A03295 image captions); M03295 shows IHC in human tonsil and colon carcinoma and IF/ICC in transfected HEK293 cells and human tissue (M03295 image captions). M03295-1 shows IHC in formaldehyde-fixed untransfected and transfected 293T cell sections after EDTA pH 9 retrieval (M03295-1 IHC caption).
Which to pick: For human tissue IHC, choose the monoclonal M03295, illustrated in tonsil and colon carcinoma at 2 μg/mL; the captions do not report a fixative (M03295 catalog and IHC captions). For human IF/ICC, M03295 has tissue IF and transfected HEK293 cell IF/ICC images, with 20 μg/mL listed for IF (M03295 catalog and IF captions). For human and mouse reactivity, A03295 lists IHC-P and IF, with IHC illustrated in formaldehyde-fixed paraffin mouse brain; M03295-1 is a human-reactive monoclonal illustrated for IHC-P in formaldehyde-fixed 293T cell sections (A03295 catalog and IHC caption; M03295-1 catalog and IHC caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q9BQ51 (PD1L2_HUMAN, Programmed cell death 1 ligand 2).
- Human Protein Atlas. PDCD1LG2 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. PDCD1LG2 subcellular location (ICC-IF): Localized to the cytosol..
- Human Protein Atlas. PDCD1LG2 antibody validation summary (1 antibodies).
- Tumor PDCD1LG2 (PD-L2) Expression and the Lymphocytic Reaction to Colorectal Cancer. Cancer immunology research 2017 — PMC5668177.
- Fusobacterium nucleatum in Colorectal Cancer Relates to Immune Response Differentially by Tumor Microsatellite Instability Status. Cancer immunology research 2018 — PMC6215508.
- Expression of programmed cell death 1 ligand 2 (PD-L2) is a distinguishing feature of primary mediastinal (thymic) large B-cell lymphoma and associated with PDCD1LG2 copy gain. The American journal of surgical pathology 2014 — PMC4994970.
- Closed system RT-qPCR as a potential companion diagnostic test for immunotherapy outcome in metastatic melanoma. Journal for immunotherapy of cancer 2019 — PMC6751819.
- PubMed PMID:11283156 — UniProt-cited evidence.
- PubMed PMID:11224527 — UniProt-cited evidence.
- PubMed PMID:15253154 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for PDCD1LG2 (Programmed cell death 1 ligand 2). Expected localisation Observed cytoplasmic (HPA tissue IHC); membrane and secreted forms annotated (UniProt). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet A03295). Validated IHC antibody: A03295 at 2.5 μg/mL (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections. Protocol steps: 1. Sample: Formaldehyde-fixed, paraffin-embedded mouse brain tissue (datasheet A03295); 2. Fixation: Image formalin-fixed; duration unreported (datasheet A03295); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat retrieval: Citrate pH 6 (datasheet A03295); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% serum (datasheet A03295); 8. Primary antibody: Rabbit anti-PDCD1LG2, 2.5 μg/mL (datasheet A03295); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formaldehyde fixation consistent (datasheet A03295 IHC) (selected-SKU IHC image A03295); Caveat: A secreted variant may weaken RNA–protein agreement (HPA tissue IHC); Isoform / epitope: 3 isoforms; check extracellular versus cytoplasmic epitope coverage (UniProt); Regulation: Staining levels vary by tissue (HPA tissue IHC). Evidence: UniProt Q9BQ51, HPA tissue IHC · Approved, HPA ICC-IF · cytosol., Datasheet A03295, PMC5668177, PMC6215508. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/PDCD1LG2/immunohistochemistry-ihc-guide.