PDCD1LG2 / Programmed cell death 1 ligand 2 · IHC design guide

Design Immunohistochemistry for PDCD1LG2

Plan human chromogenic IHC-P with M03295 at 2 μg/mL, as shown in tonsil and colon carcinoma images (datasheet M03295). Interpret variable cytoplasmic staining alongside the reported secreted variant (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for PDCD1LG2 (IHC for PDCD1LG2): expected localisation Observed cytoplasm (HPA tissue IHC); membrane form (UniProt), antibody A03295, validated IHC image, and IHC protocol steps — Printable **PDCD1LG2** IHC protocol sheet — expected localisation Observed cytoplasm (HPA tissue IHC); membrane form (UniProt), antibody A03295, controls and protocol steps. Open the full PDCD1LG2 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

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 cytoplasm (HPA tissue IHC); membrane form (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	Formaldehyde-fixed mouse sections are documented (datasheet A03295, IHC) (selected-SKU IHC image A03295)
	Caveat	A secreted variant may separate RNA and tissue staining (HPA tissue IHC)
	Regulation	Tissue-dependent staining intensity (HPA tissue IHC)
	Isoform / epitope	3 isoforms; check epitope against extracellular and cytoplasmic regions (UniProt)

Section 1

Recommended PDCD1LG2 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol with published PDCD1LG2 staining in colorectal carcinoma and lymphoma sections (PMC5668177; PMC6215508; PMC4994970).

Recommended immunohistochemistry (IHC-P) protocol parameters
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 heat retrieval for the catalog antibody (datasheet A03295); a published colorectal protocol used pH 8 retrieval (PMC5668177).

Section 2

What Is the Expected PDCD1LG2 Staining Pattern?

PDCD1LG2 is a cell membrane, endomembrane and secreted protein with an extracellular domain and a cytoplasmic tail (UniProt Q9BQ51 topology). In paraffin-section IHC, HPA reports variable cytoplasmic staining across tissues, including high staining in selected epithelial cells, cardiomyocytes and kidney tubule cells (HPA tissue IHC). HPA rates its tissue IHC evidence Approved, while noting low consistency between antibody staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Clear cytoplasmic staining in bronchial respiratory epithelial cells or kidney tubule cells.	This matches cell types scored High by HPA and supports an interpretable positive control (HPA tissue IHC). Compare the stain with tissue structure and the negative control before assigning a result (standard IHC practice).
Predominantly nuclear staining, especially without a cytoplasmic pattern.	A nuclear-only pattern is unsupported by the reported HPA tissue IHC profile and UniProt locations (HPA tissue IHC; UniProt Q9BQ51). Review control sections and detection background before treating it as PDCD1LG2 signal (standard IHC practice).
Strong staining confined to adipocytes in adipose tissue.	HPA reports PDCD1LG2 as Not detected in adipocytes (HPA tissue IHC). Check for cross-reactivity or endogenous detection activity; a stain in this cell type alone does not establish target expression (standard IHC practice).
Diffuse chromogen across tissue spaces, edges or many unrelated structures.	An unstructured deposit cannot establish the cell-associated cytoplasmic pattern reported by HPA (HPA tissue IHC). Compare a negative control and inspect blocking, washes and detection steps for nonspecific signal (standard IHC practice).
No visible stain in a high-staining reference tissue, such as bronchus or colon.	HPA scores bronchial respiratory epithelial cells and colonic glandular cells High (HPA tissue IHC). An absent reference signal makes a negative test section inconclusive; verify tissue integrity, antibody use and detection with controls (standard IHC practice).

💡Expected PDCD1LG2 appearanceCall a section positive when identifiable cells show cytoplasmic staining consistent with the tissue pattern, potentially strong in HPA High cell types; isolated nuclear stain, diffuse deposit or adipocyte-only stain is suspect (HPA tissue IHC; standard IHC practice).

How each factor affects the staining
Tissue and cell selection	HPA scores glandular cells in appendix, colon and duodenum, bronchial respiratory epithelium, cardiomyocytes and kidney tubule cells High; adipocytes are Not detected (HPA tissue IHC). Choose a reference by cell type, rather than assuming uniform staining throughout a section (standard IHC practice).
Protein topology and epitope	UniProt places residues 20–220 extracellularly, 221–241 across the membrane and 242–273 in the cytoplasmic tail (UniProt Q9BQ51 topology). The antibody epitope is unspecified here, so topology alone cannot predict its IHC compartment or retrieval response.
Processing and variants	UniProt lists a signal peptide at residues 1–19, 3 isoforms and a secreted location (UniProt Q9BQ51). HPA notes that at least one variant is secreted, complicating RNA-to-protein location comparisons; these records do not identify which variant produces a given stain (HPA tissue IHC).
Evidence strength	The listed HPA antibody, HPA013411, has Approved IHC status, while HPA reports low consistency between staining and RNA expression (HPA antibodies; HPA tissue IHC). Treat cell-level morphology and controls as essential to interpretation (standard IHC practice).
IF/ICC: what location is reported?	HPA reports cytosol as the main ICC-IF location but marks it uncertain; the listed antibody's ICC status is Uncertain (HPA subcellular; HPA antibodies). Use the separate IF/ICC guide for that application; the ICC observation does not establish an IHC protocol or override tissue IHC results.
General chromogenic workflow	Antigen retrieval, blocking, washes and detection controls can affect how an IHC slide reads (standard IHC practice). Neither supplied source establishes a PDCD1LG2-specific fixation or retrieval effect; judge any workflow change against a positive tissue and negative control.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A high-reference section has no stain.	A failed reference signal can reflect a section, antibody or detection problem; bronchial respiratory epithelium is scored High by HPA (HPA tissue IHC; standard IHC practice).	Check the reference tissue and cell identity, reagent setup and detection control; then review the catalog antibody's IHC-P instructions before interpreting test sections (standard IHC practice).
The test section is weak while its positive reference stains.	HPA reports variable cytoplasmic levels across tissues, so weak staining may reflect the sampled cells; the supplied sources give no target-specific fixation explanation (HPA tissue IHC).	Score the named cell population against its tissue-specific HPA level and the run controls; record weak staining without converting it into a categorical negative (HPA tissue IHC; standard IHC practice).
Most of the slide has diffuse color.	Diffuse deposit lacks the cell-associated cytoplasmic pattern described for tissue IHC and may reflect nonspecific background (HPA tissue IHC; standard IHC practice).	Compare the negative control, inspect washes and blocking, and assess whether color follows recognizable cell boundaries or cytoplasm (standard IHC practice).
Only nuclei stain.	Neither HPA's cytoplasmic tissue profile nor UniProt's membrane, endomembrane and secreted locations support a nuclear-only assignment (HPA tissue IHC; UniProt Q9BQ51).	Check negative controls and section morphology; withhold a PDCD1LG2-positive call unless a supported cell-associated pattern is demonstrable (standard IHC practice).
Adipocytes stain strongly while expected reference cells do not.	HPA scores adipocytes Not detected and several epithelial reference populations High, making this distribution discordant with its tissue IHC record (HPA tissue IHC).	Review cross-reactivity and endogenous detection activity with appropriate controls; verify the reference cell population before assigning target-specific staining (standard IHC practice).
RNA abundance seems inconsistent with the IHC result.	HPA explicitly reports low RNA–staining consistency and notes that a secreted variant can make RNA and protein tissue locations differ (HPA tissue IHC).	Report the observed staining by tissue, cell type and compartment alongside the RNA result; avoid using RNA alone to overrule a controlled IHC observation (HPA tissue IHC; standard IHC practice).

Source: UniProt Q9BQ51 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing PDCD1LG2 staining

Immunohistochemistry Validation of PD-L2 in Mouse Brain Tissue Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-PD-L2 antibody (A03295) at 2.5 μg/ml. Tissue was fixed with formaldehyde and blocked with 10% serum for 1 h at RT; antigen retrieval was by heat mediation with a citrate buffer (pH6). Samples were incubated with primary antibody overnight at 4˚C. A goat anti-rabbit IgG H&L (HRP) at 1/250 was used as secondary. Counter stained with Hematoxylin.

A03295 · IHCImmunohistochemistry Validation of PD-L2 in Mouse Brain Tissue Immunohistochemical analysis of paraffin-embedded mouse brain tissue using an

Immunofluorescence Validation of PD-L2 in Mouse Brain Tissue Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain cells labeling PD-L2 with A03295 at 20 μg/mL, followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (red).

A03295 · IFImmunofluorescence Validation of PD-L2 in Mouse Brain Tissue Immunofluorescent analysis of 4% paraformaldehyde-fixed mouse brain cells label

Immunohistochemical analysis of PDL-2 in untransfected(left) or transfected(right) with 293T cell sections. Cell was fixed with formaldehyde; antigen retrieval was by heat mediation with a EDTA buffer (pH9. 0). Samples were incubated with primary antibody (1:25) for 1 hours at room temperature. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

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.

Immunocytochemistry of PD-L2 in transfected HEK293 cells with PD-L2 antibody at 5 μg/mL. Lower left: Immunocytochemistry in transfected HEK293 cells with control mouse IgG antibody at 5 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-251MG — PDCD1LG2 (HPA013411)

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 look for staining in glandular cells (HPA: Appendix glandular cells, High). Use adipose tissue as the negative tissue and assess adipocytes (HPA: Adipocytes, Not detected); on the appendix slide, treat cells outside the glandular compartment as internal negative comparators only where they are visibly unstained against the no-primary control.

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 a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species, immunoglobulin class and concentration, and a biological specificity control such as PDCD1LG2 knockout tissue or a validated immunizing-peptide block (standard IHC practice). Block endogenous peroxidase and check residual background in the appendix section before interpreting chromogenic staining (standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the fixative for the selected A03295 tissue-IHC caption is unreported. The caption describes heat retrieval in citrate buffer at pH 6, but does not establish whether retrieval is required for other specimens (selected A03295 tissue-IHC caption). There is no basis here to call frozen sections or IF/ICC easier; for the separate IF/ICC guide, interpret cytosolic signal cautiously because its assignment is uncertain (HPA subcellular: Cytosol, uncertain).

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 in paraffin sections by checking retrieval, compartment, cell identity and controls before comparing signal across samples (UniProt Q9BQ51; standard IHC practice).

How should I retrieve PDCD1LG2 in paraffin sections when staining is weak?

Use heat-mediated retrieval in citrate buffer at pH 6 as the first condition for the catalog antibody (datasheet A03295). The selected tissue-IHC image used this retrieval with 2.5 µg/ml primary antibody on paraffin-embedded mouse brain, so compare any change against that tested combination (datasheet A03295 caption). Check that sections remain attached and that a positive control stains before increasing retrieval intensity (standard IHC practice). If signal remains weak, trial another retrieval buffer on matched sections as a fallback, recording tissue preservation and background alongside signal (standard IHC practice).

Could fixation explain weak or uneven PDCD1LG2 staining?

Target-specific fixation sensitivity is unknown from the supplied evidence, so treat fixation as a variable to test rather than assigning PDCD1LG2 a preferred fixation time (supplied evidence). Compare matched specimens with documented fixative and processing histories, then stain them together using the same retrieval and detection conditions (standard IHC practice). If a section shows patchy staining, inspect tissue preservation and compare the patch against an internal control before changing antibody concentration (standard IHC practice). Record whether the pattern follows specimen thickness or processing boundaries, because those observations help separate preparation effects from biological distribution (standard IHC practice).

Should PDCD1LG2 staining appear at the cell surface or in cytoplasm?

Expect membrane-associated signal to be biologically plausible: PDCD1LG2 has an extracellular region spanning residues 20–220 and a transmembrane segment at 221–241 (UniProt Q9BQ51 topology). Cytoplasmic staining also warrants evaluation because the tissue-IHC profile reports variable cytoplasmic expression, while the subcellular cytosol assignment is uncertain (HPA tissue IHC; HPA subcellular). Score membranous and cytoplasmic patterns separately in morphologically identified cells, using the same criteria across sections (standard IHC practice). Treat diffuse staining outside intact cells cautiously, since at least one protein variant is secreted and RNA and protein locations may differ (HPA reliability description).

How do isoforms and epitope position affect PDCD1LG2 IHC interpretation?

PDCD1LG2 has 3 annotated isoforms, so establish which forms the antibody detects before treating staining intensity as total protein abundance (UniProt Q9BQ51 isoforms; standard IHC practice). Its extracellular region spans residues 20–220, the membrane segment 221–241, and the cytoplasmic tail 242–273 (UniProt Q9BQ51 topology). Five annotated glycosylation sites lie in the extracellular region, making documented epitope placement useful when comparing antibodies or staining patterns (UniProt Q9BQ51 glycosylation and topology; standard IHC practice). If the catalog epitope is unspecified, report compartment-specific staining without claiming isoform selectivity or glycosylation-dependent detection (standard IHC practice).

How should I assess PDCD1LG2 by IF alongside the IHC findings?

For a separate IF assessment, multiplex PDCD1LG2 with a marker identifying the cell population being scored, such as a validated epithelial marker when examining the reported respiratory epithelial signal (HPA tissue IHC; standard IF practice). Inspect unstained tissue autofluorescence and choose a fluorophore channel with clear separation from it before interpreting dim signal (standard IF practice). If the antibody epitope is extracellular, first evaluate staining without permeabilisation; an intracellular epitope requires access through the membrane (UniProt Q9BQ51 topology; standard IF practice). Because the catalog epitope is unspecified here, document the epitope and compare controls before relating an IF pattern to chromogenic IHC (supplied evidence; standard IF practice).

What should I check when PDCD1LG2 chromogenic staining is diffuse?

Start with a no-primary control and inspect whether colour persists in the same structures; persistent colour points to detection-system or tissue background (standard IHC practice). Check endogenous peroxidase blocking before HRP detection, and assess DAB development and hematoxylin counterstain on matched sections (standard chromogenic IHC practice). The selected image used 10% serum blocking for 1 hour at room temperature, followed by overnight primary incubation at 4 °C; those are documented conditions for this antibody (datasheet A03295 caption). Evaluate diffuse extracellular colour separately from cell-associated staining because a secreted variant is reported (HPA reliability description; standard IHC practice).

How can I quantify PDCD1LG2 staining across different tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION

Choose a predefined readout, such as an H-score or percentage of positive cells for a named cell population, and keep thresholds fixed across the comparison (standard IHC practice). For infiltrating cells, positive-cell density per mm² can be useful when the sampled tissue area is recorded (standard IHC practice). Normalise each readout to the relevant viable cells or tissue area, and exclude folds, edges and necrotic regions using the same rules for every section (standard IHC practice). Record membranous and cytoplasmic scores separately, since PDCD1LG2 has membrane topology and variable cytoplasmic tissue staining is reported (UniProt Q9BQ51 topology; HPA tissue IHC).

How can I distinguish convincing PDCD1LG2 positivity from artefact?

A convincing result repeats in intact, morphologically identified cells and agrees with the chosen positive control and compartment scoring rules (standard IHC practice). Membrane-associated staining fits PDCD1LG2 topology, while cytoplasmic staining needs careful assessment because HPA reports it across tissues with low consistency against RNA expression (UniProt Q9BQ51 topology; HPA tissue IHC and reliability description). Check whether an apparent signal is confined to section edges, necrosis or sites that also stain in a no-primary control (standard IHC practice). Persistent colour after omitting primary antibody can indicate endogenous enzyme or detection background, so avoid counting it as PDCD1LG2-positive cells (standard chromogenic IHC practice).

Boster reagents

Best PDCD1LG2 / Programmed cell death 1 ligand 2 IHC Antibodies

The catalog shows PDCD1LG2 IHC data from mouse brain, human tonsil, human colon carcinoma, and 293T cell sections, with IF data from mouse brain and human samples (catalog image captions).

Real IHC data

Anti-PD-L2 PDCD1LG2 Antibody

Cat # A03295

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Real IHC data Immunohistochemical analysis of PDL-2 in untransfected(left) or transfected(right) with 293T cell sections. Cell was fixed with formaldehyde; antigen retrieval was by heat mediation with a EDTA buffer (pH9. 0). Samples were incubated with primary antibody (1:25) for 1 hours at room temperature. A undiluted biotinylated goat polyvalent antibody was used as the secondary antibody.

Anti-PDCD1LG2 Antibody (N-term)

Cat # M03295-1

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Real IHC data Immunohistochemistry of PD-L2 in human tonsil tissue with PD-L2 antibody at 2 μg/mL.

Anti-PDL2 PDCD1LG2 Monoclonal Antibody [4E10]

Cat # M03295

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A03295 has IHC data from formaldehyde-fixed paraffin mouse brain and IF data from mouse brain cells and tissue (A03295 image captions). M03295 has IHC data from human tonsil and colon carcinoma and IF/ICC data from those tissues and transfected HEK293 cells (M03295 image captions); M03295-1 has IHC data from untransfected and transfected 293T cell sections (M03295-1 image caption).

Which to pick: For human tissue IHC, choose M03295, a mouse monoclonal with tonsil and colon carcinoma IHC images; its IHC captions do not report a fixative (catalog M03295; M03295 IHC captions). For IF/ICC, choose M03295 for human samples (catalog M03295 applications and IF/ICC captions); M03295-1 is an IHC-P option supported by formaldehyde-fixed 293T cell sections retrieved with EDTA at pH 9.0 (M03295-1 image caption). For human and mouse reactivity, choose rabbit antibody A03295, whose IHC image shows formaldehyde-fixed paraffin mouse brain retrieved with citrate at pH 6; its catalog also lists IF, with mouse brain IF images (catalog A03295; A03295 image captions).

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.