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HLA-DOA / HLA class II histocompatibility antigen, DO alpha chain · IHC design guide
Design Immunohistochemistry for HLA-DOA
Plan HLA-DOA IHC in paraffin sections using lymph node as a positive tissue and assessing cytoplasmic staining (HPA tissue IHC). This guide covers fixation, retrieval, controls and interpretation of staining in non-lymphoid cells (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HLA-DOA 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 | Cytoplasmic staining in several tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining, strongest in a subset of lymphoid cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30721) | |
| Positive control | Lymph node+4 more · see all | |
| Negative control | Bone marrow+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30721) | |
| Caveat | Myocytes and keratinocytes also stain strongly (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | No isoforms; epitope position across residues 26–217 vs 241–250 matters (UniProt) |
Section 1
Recommended HLA-DOA IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: A30721) and a published HLA-DOA colorectal carcinoma protocol (PMC6230233) provide staining conditions.
| Sample | Paraffin-embedded Human breast cancer tissue; fixative not specified (datasheet A30721) |
| Fixation | Image fixative and duration unreported (datasheet A30721); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 8.0 (datasheet A30721); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-HLA-DOA, 1:100 - 1:300 (datasheet A30721) |
| 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 | HLA-DOA-positive staining in non-germinal center cells of lymph node (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues, most abundant in a subset of lymphoid cells. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 8.0 heat retrieval (datasheet: A30721). The published alternative used basic CC1 at 95 °C for 56 min (PMC6230233).
Section 2
What Is the Expected HLA-DOA Staining Pattern?
Expect predominantly cytoplasmic staining in a subset of lymphoid cells (HPA: tissue IHC, Enhanced reliability), consistent with HLA-DOA on endosomal and lysosomal membranes (UniProt P06340: subcellular location). Its single transmembrane segment spans residues 218–240 (UniProt P06340: topology). HPA reports high staining in non-germinal center cells of lymph node and tonsil and in splenic white pulp cells (HPA: tissue IHC).
| Strong, localized cytoplasmic signal in non-germinal center cells of lymph node or tonsil, or cells in splenic white pulp (HPA: High). | This matches the reported tissue pattern (HPA: tissue IHC). A granular or punctate component is plausible for an endosomal or lysosomal membrane protein (UniProt P06340: subcellular location), although HPA does not specify that appearance. |
| Uniform nuclear staining, or a compartment pattern unrelated to cytoplasm or membranes. | Treat it as an unexpected pattern and investigate staining artefact: HPA describes cytoplasmic tissue staining, while UniProt places HLA-DOA on endosomal and lysosomal membranes (HPA: tissue IHC; UniProt P06340: subcellular location). |
| Strong staining in a cell population reported as not detected, such as bone-marrow hematopoietic cells (HPA: Not detected). | Check for antibody cross-reactivity or endogenous detection activity (general IHC practice). Cell identity matters: high staining is also reported in skeletal-muscle myocytes and skin keratinocytes (HPA: tissue IHC), so non-lymphoid staining alone is not discordant. |
| Diffuse chromogen across cells and surrounding tissue, without a discernible cell or compartment pattern. | This is difficult to score as HLA-DOA: the reference pattern is selective cytoplasmic staining (HPA: tissue IHC). Review background in a no-primary control and the blocking and detection steps (general IHC practice). |
| No signal in lymph-node non-germinal center cells or splenic white pulp cells (HPA: High). | A failed positive control is possible, but absence alone does not identify the cause (general IHC practice). Confirm tissue landmarks and control performance before interpreting a test section as negative (general IHC practice). |
💡Expected HLA-DOA appearanceCall a positive result when staining is concentrated in the cytoplasm of expected cells, especially strongly stained non-germinal center lymphoid cells (HPA: High; HPA: cytoplasmic expression); uniform nuclear or diffuse tissue-wide color is an unexpected pattern (HPA: tissue IHC; UniProt P06340: subcellular location).
| Tissue and cell selection (HPA: tissue IHC) | Lymph node, tonsil and spleen provide reported high-staining populations; compare the named cell populations, since a whole-tissue positive or negative label can obscure the relevant result (HPA: High). |
| Reference-pattern confidence (HPA: Enhanced reliability) | HPA calls tissue staining Enhanced, but describes only medium consistency with RNA data (HPA: reliability description). Use the reported cell pattern as a reference while checking uncertain results with controls (general IHC practice). |
| Antibody validation (HPA: antibody records) | HPA045038 is IHC Enhanced and HPA076922 is IHC Supported (HPA: antibody records). These ratings describe validation status; they do not establish that every stain from either antibody is target-specific. |
| Topology and processing (UniProt P06340) | The mature chain begins at residue 26; residues 218–240 form the membrane span (UniProt P06340: processing, topology). The supplied evidence gives no antibody epitope, so it cannot predict epitope-specific retrieval needs. |
| Glycosylation (UniProt P06340: PTMs) | Sites at residues 104 and 144 are annotated (UniProt P06340: glycosylation). Their presence does not establish an effect on this antibody's staining or antigen retrieval. |
| IF/ICC expectation? | HPA summarizes the subcellular location as membrane, but lists no main location or ICC-IF image cell lines (HPA: subcellular). UniProt supports an endosomal or lysosomal membrane interpretation; an IF/ICC image pattern is unverified here (UniProt P06340: subcellular location). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected positive cells show no chromogenic signal (HPA: High in lymph-node non-germinal center cells). | The assay or section may have failed; the absence of signal does not identify which step failed (general IHC practice). | Run an expected-positive section alongside the test, then check primary-antibody application, detection reagents and counterstain visibility (general IHC practice). |
| Signal is weak in an expected high-staining population (HPA: High in splenic white pulp cells). | Weak staining could reflect assay conditions or sampling of a different cell population (general IHC practice). | Confirm the cell population on the counterstained section and compare control performance before adjusting the IHC workflow (general IHC practice). |
| The slide has diffuse background that obscures cell boundaries. | Nonspecific binding, inadequate blocking or detection background can produce widespread color (general IHC practice). | Examine a no-primary control, then review blocking, washes and chromogen development (general IHC practice). |
| Nuclei stain strongly while the expected cytoplasmic pattern is absent (HPA: cytoplasmic expression). | The compartment is discordant with HPA tissue staining and UniProt membrane localization; staining artefact is a possibility (HPA: tissue IHC; UniProt P06340: subcellular location). | Compare with an expected-positive section and a no-primary control; inspect the counterstain and detection steps (general IHC practice). |
| A reported negative cell population stains strongly, such as bone-marrow hematopoietic cells (HPA: Not detected). | Cross-reactivity or endogenous detection activity is possible (general IHC practice); the HPA result applies to the named cell population (HPA: tissue IHC). | Confirm cell identity, examine a no-primary control and compare staining with an expected-positive population (general IHC practice). |
| Staining appears in myocytes or keratinocytes and seems inconsistent with a lymphoid target (HPA: High in both). | Those populations are reported as high-staining in HPA tissue IHC (HPA: skeletal muscle myocytes; skin keratinocytes). | Score the identified cell population and its compartment against the corresponding HPA reference; do not classify these cells as unexpected solely because they are non-lymphoid (HPA: tissue IHC). |
Experiment evidence
IHC & IF images showing HLA-DOA staining
A30721 · IHCImmunohistochemical analysis of paraffin-embedded Human breast cancer. Antibody was diluted at 1:100(4° overnight). High-pressure and temper
A06776-1 · IHCImmunohistochemistry (IHC) analyzes of HLA-DOα (V111) pAb in paraffin-embedded human breast carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000204252-HLA-DOA/tissue/cerebral+cortex#imid_12209853
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000204252-HLA-DOA/tissue/colon#imid_12202367
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000204252-HLA-DOA/tissue/liver#imid_12202376
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000204252-HLA-DOA/tissue/kidney#imid_12202361
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HLA-DOA IHC & IF
🧪Run lymph node first and expect staining in non-germinal center cells (HPA: High); use bone marrow hematopoietic cells as the negative tissue (HPA: Not detected). On the positive slide, cells outside the stained compartment should show only counterstain or background, but their identity as true negatives requires validation because the supplied HPA row specifies only non-germinal center cells (HPA: lymph node row).
Positive control tissue: Lymph node (Non-germinal center cells, HPA High)
Negative control tissue: Bone marrow (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for HLA-DOA; derive a cell-line control from the positive tissue's cell type (Non-germinal center cells) and confirm it by RNA or western blot first.
Technical controls: Include no-primary or secondary-only and primary-matched isotype controls, matching the primary antibody’s host species and immunoglobulin class; use knockout tissue or immunogen-peptide blocking as a biological specificity control (selected-SKU caption: peptide-blocked control). For chromogenic lymph-node IHC, quench endogenous peroxidase and check for residual background (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected-SKU paraffin-section caption does not state its fixative (selected-SKU caption). The caption uses high-pressure, high-temperature Tris–EDTA retrieval at pH 8.0, but does not establish that retrieval is required (selected-SKU caption). Neither frozen sections nor IF are established as easier by the supplied evidence; for lymph node, endogenous peroxidase can complicate chromogenic interpretation (standard IHC practice).
HPA tissue IHC evidence for HLA-DOA
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data.). 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 |
|---|---|---|---|---|
| Lymph node | Non-germinal center cells | High | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | High | Protein (IHC) | HPA → |
| Skin | Keratinocytes | High | Protein (IHC) | HPA → |
| Spleen | Cells in white pulp | High | Protein (IHC) | HPA → |
| Tonsil | Non-germinal center cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HLA-DOA IHC Tips
Troubleshoot HLA-DOA staining in chromogenic IHC of paraffin sections using the catalog antibody’s retrieval conditions and compartment-specific controls.
How should I retrieve HLA-DOA antigen in paraffin sections?
Start with heat-mediated retrieval in Tris-EDTA at pH 8.0 for paraffin sections (datasheet A30721). The selected tissue image used high pressure and temperature with this buffer, but its caption does not report a heating duration (caption A30721). For an initial run, hold sections at retrieval temperature for 15–20 minutes, cool them for 20 minutes, and keep those timings identical across controls (standard IHC practice). If staining is weak, adjust heating time in a small series before testing another retrieval buffer, because excessive heating can damage section morphology (standard IHC practice). Compare each condition against a positive lymphoid compartment and a primary-antibody-omission control (HPA tissue IHC; standard IHC practice).
Could fixation explain weak HLA-DOA staining?
The selected image identifies paraffin-embedded human breast cancer but does not state its fixative, so target-specific fixation sensitivity is unknown (caption A30721). For a controlled comparison, record tissue thickness and fixation duration; 10% neutral buffered formalin for roughly 6–24 hours is a general starting range for routine tissue processing, not an HLA-DOA-specific condition (standard IHC practice). Keep retrieval at Tris-EDTA pH 8.0 while comparing differently processed sections (datasheet A30721; standard IHC practice). Include a positive tissue section processed alongside the specimen so a staining loss can be assessed against processing variation (HPA tissue IHC; standard IHC practice). Do not infer fixation tolerance from tissue staining patterns or protein topology (HPA tissue IHC; UniProt P06340 topology).
Where should convincing HLA-DOA staining appear?
Assess chromogenic signal primarily within cells, with attention to a vesicular or membrane-associated cytoplasmic pattern: HLA-DOA is assigned to endosome and lysosome membranes (UniProt P06340 subcellular location). HPA describes cytoplasmic staining in several tissues, most abundant in a subset of lymphoid cells (HPA tissue IHC). The protein has one membrane-spanning segment at residues 218–240 and a short cytoplasmic tail at 241–250, so staining need not outline the entire cell surface (UniProt P06340 topology). Review signal at higher magnification against cell boundaries and the counterstain before scoring it as intracellular (standard IHC practice). Treat diffuse extracellular deposit or uniform nuclear color as a reason to inspect controls and detection chemistry (UniProt P06340 subcellular location; standard IHC practice).
How does the unknown epitope affect IHC optimisation?
The supplied record lists 0 isoforms and does not locate the catalog antibody’s epitope, so an isoform-specific staining claim is unsupported (UniProt P06340 record; supplied antibody evidence). The mature chain spans residues 26–250 after removal of the 1–25 signal peptide (UniProt P06340 processing). Its 26–217 extracellular region includes glycosylation sites at 104 and 144, while residues 241–250 form the cytoplasmic tail (UniProt P06340 topology and glycosylation). Because the recognised region is unspecified, compare retrieval conditions empirically rather than attributing a weak section to glycosylation or epitope masking (supplied antibody evidence; standard IHC practice). Peptide preabsorption reduced staining in the selected image, but interpret that control alongside tissue localisation and omission controls (caption A30721; standard IHC practice).
How can I check the IHC pattern by multiplex IF?
Use multiplex IF as a separate localisation check, pairing HLA-DOA with a B-cell marker such as CD20 because HLA-DOA interacts with HLA-DM in B cells (UniProt P06340 function and subunit; standard IF practice). Choose a far-red channel for the weaker target signal and inspect unstained tissue in each channel to gauge autofluorescence (standard IF practice). HLA-DOA’s large domain is on the extracellular side of the membrane, which faces the lumen of intracellular endosomes and lysosomes; detergent access may therefore matter for an antibody recognising that domain (UniProt P06340 topology and subcellular location; standard IF practice). As the catalog antibody’s epitope is unspecified, compare mild permeabilisation, such as 0.1% Triton X-100 for 5 minutes, against an untreated section (supplied antibody evidence; standard IF practice). Keep single-stain controls for channel bleed-through when comparing IF with chromogenic IHC (standard IF practice).
What should I change when the DAB background obscures HLA-DOA?
First inspect a primary-antibody-omission section for signal produced by the detection system, including endogenous peroxidase activity (standard IHC practice). A 3% hydrogen peroxide block for about 10 minutes, followed by a protein block for 20–30 minutes, is a general chromogenic IHC starting workflow (standard IHC practice). The selected image used the catalog antibody at 1:100 overnight at 4°C; titrate concentration and incubation if diffuse color persists after detection controls are clean (caption A30721; standard IHC practice). Wash thoroughly between antibody and detection steps and compare DAB development times on matched sections (standard IHC practice). The caption’s peptide-preabsorbed negative control supports an antibody-dependent component in that image but does not replace controls for endogenous enzyme or nonspecific secondary binding (caption A30721; standard IHC practice).
How should I score heterogeneous HLA-DOA staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the anatomical compartment and eligible cell population before counting, because HPA reports strongest cytoplasmic expression in a subset of lymphoid cells (HPA tissue IHC; standard IHC practice). Report the percentage of positive eligible cells and an H-score from 0–300, using the same intensity thresholds across slides (standard IHC practice). If spatial abundance matters, count positive cells per mm² of the selected compartment and state how fields were sampled (standard IHC practice). Normalise cell counts to the number of eligible cells or measured compartment area, rather than treating variable lymphoid content as a staining change (HPA tissue IHC; standard IHC practice). Set the positivity threshold using matched negative controls, and assess faint cytoplasmic color consistently (standard IHC practice).
How can I distinguish true HLA-DOA signal from artefact?
Give greatest weight to intracellular staining in the relevant cell population: HLA-DOA associates with endosome and lysosome membranes, and HPA reports abundant cytoplasmic signal in a subset of lymphoid cells (UniProt P06340 subcellular location; HPA tissue IHC). Lymph node non-germinal center cells and spleen white-pulp cells have reported high staining, making them useful comparison compartments (HPA tissue IHC). Uniform nuclear staining, extracellular DAB precipitate, or color confined to section edges warrants review against topology and processing controls (UniProt P06340 topology; standard IHC practice). Exclude necrotic regions from scoring and examine a primary-antibody-omission section for endogenous enzyme signal (standard IHC practice). Interpret the selected image’s peptide-preabsorbed control as supporting antibody-dependent staining in that specimen, without treating it as proof that every stained cell expresses HLA-DOA (caption A30721; standard IHC practice).
Boster reagents
Best HLA-DOA / HLA class II histocompatibility antigen, DO alpha chain IHC Antibodies
Two anti-HLA-DOA antibodies have IHC images from paraffin-embedded human breast cancer or carcinoma (catalog image captions). Human IHC is listed for both; IF/ICC is listed for A30721, and mouse and rat reactivity for A06776-1 (catalog applications/reactivity).
Real IHC data
Anti-HLA-DOA/Hla Doa Antibody
Cat # A30721
Real IHC data
Anti-HLA-DO Alpha (V111) HLA-DOA Antibody
Cat # A06776-1
A30721 will render with a human breast cancer IHC image showing paraffin sections, 1:100 antibody dilution, overnight incubation at 4°C and Tris-EDTA retrieval at pH 8.0 (A30721 image caption). A06776-1 will render with a paraffin-embedded human breast carcinoma IHC image at 1:100 (A06776-1 image caption).
Which to pick: For tissue IHC, either SKU has a paraffin-section image; A30721 also documents retrieval and incubation conditions in its caption (catalog image captions). Choose A30721 for IF/ICC because those applications are listed for it; its figure documents IHC only (A30721 applications/image caption). Consider A06776-1 when mouse or rat reactivity is needed, while recognizing that its pictured IHC sample is human; fixation is unreported in both captions (catalog reactivity/image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P06340 (DOA_HUMAN, HLA class II histocompatibility antigen, DO alpha chain).
- Human Protein Atlas. HLA-DOA tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HLA-DOA subcellular location (ICC-IF): Membrane.
- Human Protein Atlas. HLA-DOA antibody validation summary (2 antibodies).
- Two histologically colorectal carcinomas subsets from the serrated pathway show different methylome signatures and diagnostic biomarkers. Clinical epigenetics 2018 — PMC6230233.
- PubMed PMID:3000765 — UniProt-cited evidence.
- PubMed PMID:2499532 — UniProt-cited evidence.
- PubMed PMID:2370084 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HLA-DOA (HLA class II histocompatibility antigen, DO alpha chain). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30721). Validated IHC antibody: A30721 at 1:100 - 1:300 (the product's own IHC validation image is shown). Controls (HPA): positive Lymph node (Non-germinal center cells); negative Bone marrow. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded Human breast cancer tissue; fixative not specified (datasheet A30721); 2. Fixation: Image fixative and duration unreported (datasheet A30721); 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: Tris-EDTA pH 8.0 (datasheet A30721); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-HLA-DOA, 1:100 - 1:300 (datasheet A30721); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A30721); Caveat: Myocytes and keratinocytes also stain strongly (HPA tissue IHC); Isoform / epitope: No isoforms; epitope position across residues 26–217 vs 241–250 matters (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt P06340, HPA tissue IHC · Enhanced, HPA ICC-IF · Membrane, Datasheet A30721, PMC6230233. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HLA-DOA/immunohistochemistry-ihc-guide.