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HBA1 / Hemoglobin subunit alpha · IHC design guide
Design Immunohistochemistry for HBA1
Use bone marrow erythrocyte staining as a reference for chromogenic HBA1 IHC in paraffin sections (HPA tissue IHC). The guide covers staining controls and interpretation where the catalog antibody also recognizes HBA2 (catalog A00233-1).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HBA1 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 | Erythrocyte positivity; intracellular compartment unassigned (HPA tissue IHC) | |
| Staining pattern | Distinct erythrocyte staining in bone marrow (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A00233-1) | |
| Positive control | Bone marrow+1 more · see all | |
| Negative control | Adipose tissue+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific); target-specific effects are unknown. | |
| Caveat | HBA2 recognition may confound HBA1 attribution (catalog A00233-1; HPA tissue IHC) | |
| Regulation | Staining intensity reflects erythrocyte content (UniProt; HPA tissue IHC) | |
| Isoform / epitope | 0 isoforms reported; mature chain spans residues 2–142 (UniProt) |
Section 1
Recommended HBA1 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (datasheet: A00233-1) with two published chromogenic IHC protocols (PMC7753232; PMC3547883).
| Sample | Paraffin-embedded human placenta tissue; fixative not specified (datasheet A00233-1) |
| Fixation | Image fixative and duration unreported (datasheet A00233-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A00233-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A00233-1) |
| Primary antibody | Rabbit anti-HBA1, 2-5μg/ml (datasheet A00233-1) |
| Primary incubation | Overnight at 4 °C (datasheet A00233-1) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A00233-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HBA1-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Selective expression in bone marrow with distinct positivity in erythrocytes. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval (datasheet: A00233-1; PMC7753232). Try citrate pH 6.0 if needed (PMC3547883).
Section 2
What Is the Expected HBA1 Staining Pattern?
HBA1 is associated with red blood cells (UniProt P69905: tissue specificity). In paraffin sections, expect intracellular staining of erythrocytes, particularly in bone marrow, and staining in splenic red pulp (HPA: distinct erythrocyte positivity in bone marrow; High in red pulp cells). UniProt does not annotate a subcellular location, but reports no transmembrane segment (UniProt P69905: topology). HPA rates the tissue pattern Enhanced while cautioning that staining may reflect protein from more than one gene (HPA: reliability description).
| Strong staining outlines the contents of erythrocytes in bone marrow; nearby hematopoietic cells may also stain. | This fits the reported marrow pattern: High staining in hematopoietic cells with distinct erythrocyte positivity (HPA: bone marrow tissue IHC). Score the cell population and distribution together; color intensity alone cannot establish which alpha-globin gene supplied the protein (HPA: reliability description). |
| Cells in splenic red pulp stain strongly, while adipocytes in adipose tissue lack comparable staining. | The contrast agrees with High staining in splenic red pulp cells and Not detected staining in adipocytes (HPA: spleen and adipose tissue IHC). Red pulp staining is supportive context, but its cell identity should be checked morphologically before assigning a result to erythrocytes (HPA: red pulp cell designation; general IHC practice). |
| A crisp nuclear, membrane-only, or extracellular signal dominates where erythrocytes are present. | Treat the compartment assignment as suspect: hemoglobin alpha is a red-blood-cell protein with no annotated membrane-spanning segment or secretory signal (UniProt P69905: tissue specificity, topology, processing). Review morphology and controls before reporting localization; UniProt does not provide a formal subcellular annotation for HBA1 (UniProt P69905: subcellular location). |
| Adrenal glandular cells, bronchial respiratory epithelium, or adipocytes stain prominently. | Those cell types are reported as Not detected (HPA: adrenal gland, bronchus, adipose tissue IHC). Check for antibody cross-reactivity and detection-system activity with appropriate controls (general IHC practice). Do not call such staining HBA1-specific on appearance alone, especially given HPA's more-than-one-gene caution (HPA: reliability description). |
| Brown color spreads across the section, or bone marrow has no identifiable positive erythrocytes. | Widespread color obscures the reported cell pattern; an absent marrow signal conflicts with its High staining and distinct erythrocyte positivity (HPA: bone marrow tissue IHC). Interpret either result only after checking section quality, detection controls, and whether erythrocytes are actually present in the sampled area (general IHC practice). |
💡Expected HBA1 appearanceA convincing positive is strong intracellular color in morphologically identified marrow erythrocytes, with staining also expected in splenic red pulp cells (HPA: bone marrow and spleen, High); dominant nuclear color or prominent staining of HPA Not detected cell types is suspect (UniProt P69905: topology; HPA: negative tissues).
| Tissue and cell selection | Bone marrow offers distinct erythrocyte positivity, and spleen offers High red pulp staining (HPA: tissue IHC). Adipocytes and several listed glandular or epithelial cell types are Not detected comparators (HPA: negative tissues). Compare identified cells within each section rather than treating all tissue-wide color as target signal (general IHC practice). |
| Location and protein processing | The annotated chain spans residues 2–142, with no signal peptide, propeptide, or transmembrane segment (UniProt P69905: processing, topology). Those annotations support examining intracellular erythrocyte contents; they do not supply a validated microscopic subcellular location or establish how fixation affects staining (UniProt P69905: subcellular location; HPA: tissue IHC). |
| Antibody interpretation | HPA lists CAB038417 as IHC Enhanced and HPA043780 as IHC Supported (HPA: antibody validation). HPA also cautions that its tissue staining targets protein from more than one gene (HPA: reliability description). A matching pattern supports an alpha-globin-associated result, but morphology and controls cannot by themselves prove gene-specific HBA1 detection (HPA: reliability description; general IHC practice). |
| Molecular variation | UniProt lists 0 isoforms and multiple modified or glycosylated sites (UniProt P69905: isoforms, modifications, glycosylation). No antibody epitope or target-specific fixation study is supplied, so these annotations cannot predict a particular retrieval requirement, staining loss, or alternate compartment (UniProt P69905: annotations; HPA: supplied tissue and antibody records). |
| Chromogenic detection background | Erythrocyte-rich areas require careful review of chromogen controls because endogenous activity or background color can complicate peroxidase-based IHC interpretation (general IHC practice). Evaluate staining against a no-primary control and the expected cell distribution; the supplied HPA tissue pattern does not establish the source of background in an individual section (HPA: tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Bone marrow is blank despite visible erythrocytes. | The observed result conflicts with HPA's distinct, High marrow erythrocyte-associated pattern (HPA: bone marrow tissue IHC); the cause is not determined by that record. | Confirm the section contains intact erythrocytes, then check the catalog antibody's stated IHC-P conditions, detection reagents, and a positive control (general IHC practice). Do not infer an HBA1-specific fixation defect without evidence. |
| Splenic red pulp is positive, but the stained cells are unclear. | HPA reports High staining in red pulp cells without assigning every stained cell to a named lineage (HPA: spleen tissue IHC). | Identify cells from morphology and counterstain before scoring them; compare the distribution with a marrow section showing distinct erythrocyte positivity (HPA: bone marrow tissue IHC; general IHC practice). |
| Nuclei or tissue edges carry the strongest color. | That pattern does not match an intracellular erythrocyte-centered reading of the reported tissue distribution (HPA: bone marrow tissue IHC; UniProt P69905: red-blood-cell specificity). | Inspect a no-primary control and section edges, then adjust detection or blocking using ordinary IHC controls if background persists (general IHC practice). Avoid assigning HBA1 a nuclear location from this result. |
| Adipocytes or respiratory epithelial cells stain strongly. | Those cells are Not detected in the supplied HPA tissue IHC data (HPA: adipose tissue and bronchus). Cross-reactivity or detection background is possible (general IHC practice). | Compare matched negative tissue and no-primary controls, then reassess antibody specificity. HPA warns that the tissue stain may recognize protein from more than one gene (HPA: reliability description). |
| A broad brown haze prevents cell-level scoring. | Diffuse color cannot be matched reliably to the distinct marrow erythrocyte pattern (HPA: bone marrow tissue IHC); its source needs experimental controls (general IHC practice). | Check no-primary and detection-only controls, blocking, washing, and chromogen development, then score only resolvable cells (general IHC practice). Do not interpret haze as evidence of widespread HBA1 expression. |
| Can this IHC-P pattern be used as an ICC/IF localization result? | HPA supplies no ICC-IF images or main subcellular location for HBA1, and neither listed antibody has an ICC validation entry (HPA: subcellular and antibody records). | Use the separate IF/ICC guide for that application. Treat the paraffin-section tissue pattern as IHC evidence only; it does not establish an ICC/IF compartment or an IF protocol (HPA: tissue, subcellular, and antibody records). |
Experiment evidence
IHC & IF images showing HBA1 staining
A00233-1 · IHCIHC analysis of Hemoglobin using anti-Hemoglobin antibody (A00233-1). Hemoglobin was detected in a paraffin-embedded section of human placen
A00233-1 · IHCIHC analysis of Hemoglobin using anti-Hemoglobin antibody (A00233-1). Hemoglobin was detected in a paraffin-embedded section of human tonsil
M00233 · IHCImmunohistochemical analysis of paraffin-embedded human spleen, using hemoglobin subunit alpha Antibody.
M00233 · IHCImmunohistochemical analysis of paraffin-embedded mouse embryo tissue using anti-Hemoglobin subunit alpha antibody. The section was pre-trea
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000188536-HBA1/tissue/cerebral+cortex#imid_12616838
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000188536-HBA1/tissue/colon#imid_12599463
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000188536-HBA1/tissue/liver#imid_12599480
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000188536-HBA1/tissue/kidney#imid_12599473
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HBA1 IHC & IF
🧪Run bone marrow first and score staining in hematopoietic cells (HPA: High in bone marrow hematopoietic cells). Use adipose tissue as the negative and score adipocytes, which are listed as Not detected (HPA: adipocytes Not detected); on the bone marrow slide, use morphologically nonerythroid cells as an internal background comparison, confirming their staining rather than assuming they are negative (UniProt P69905: tissue specificity in red blood cells).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for HBA1; derive a cell-line control from the positive tissue's cell type (Hematopoietic cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control, a concentration-matched nonimmune rabbit IgG isotype control, and a verified HBA1 knockout specimen or validated peptide-block control (selected-SKU caption: rabbit primary and anti-rabbit secondary). Block endogenous peroxidase and assess whether blood-associated color obscures DAB staining in bone marrow (HPA: High in bone marrow hematopoietic cells; UniProt P69905: tissue specificity in red blood cells).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact A00233-1 paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used in that caption, but whether retrieval is required is unreported (selected-SKU caption). Neither frozen sections nor IF can be judged easier from the supplied evidence; bone marrow blood content may complicate assignment of hemoglobin staining to individual cells (HPA subcellular: no ICC-IF images; HPA: High in bone marrow hematopoietic cells; UniProt P69905: tissue specificity in red blood cells).
HPA tissue IHC evidence for HBA1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene.). 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 |
|---|---|---|---|---|
| Bone marrow | Hematopoietic cells | High | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HBA1 IHC Tips
Troubleshoot HBA1 staining in paraffin sections by checking retrieval, cell identity, assay controls, and the specificity limits of hemoglobin antibodies.
What retrieval should I start with for weak HBA1 staining in paraffin sections?
Start with heat-mediated retrieval in EDTA, pH 8.0 (datasheet A00233-1). The selected paraffin-section image used that condition, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A00233-1). If staining is weak, vary heating time in small steps while keeping a positive section and its processing constant; excessive heating can damage morphology and increase background (standard IHC practice). Compare erythrocyte-rich areas across runs, and assess both signal and tissue preservation before changing antibody concentration (HPA: distinct erythrocyte positivity; standard IHC practice).
Could fixation explain weak or uneven HBA1 staining?
Target-specific sensitivity to fixation is unknown: the selected paraffin-section caption does not state a fixative (datasheet A00233-1). Record the fixative, exposure time, tissue thickness, and time before fixation for each specimen, then compare sections processed under matched conditions (standard IHC practice). If signal varies, evaluate a consistently processed positive control alongside each batch and change only one processing variable at a time (standard IHC practice). Do not attribute a difference to HBA1 modification or membrane accessibility without direct evidence; assess morphology, retrieval consistency, and erythrocyte staining before assigning a cause (UniProt P69905: modified residues and no transmembrane segment; HPA: erythrocyte positivity).
Where should convincing HBA1 staining appear in tissue?
Expect staining to track erythrocytes, including those in vessel lumina, because HBA1 is an alpha-globin component of hemoglobin and is expressed in red blood cells (UniProt P69905: subunit and tissue specificity; HPA: distinct erythrocyte positivity). The record supplies no annotated subcellular location, so do not treat a particular intracellular pattern as independently validated (UniProt P69905: subcellular location not annotated). Review the chromogen against a counterstain and vessel outlines to distinguish cells from pigment or extracellular deposits (standard IHC practice). Diffuse staining in unrelated nucleated cells needs matched controls and independent evidence before being assigned to HBA1 (HPA: selective erythrocyte expression; standard IHC practice).
Can this stain distinguish HBA1 from other hemoglobin signals?
Treat this assay as hemoglobin immunoreactivity unless antibody specificity establishes more: the selected product is described as an anti-Hemoglobin antibody, and the HPA reliability note warns that staining can target protein from more than one gene (datasheet A00233-1; HPA: reliability description). The record lists zero HBA1 isoforms and places the globin domain at residues 2–142, but supplies no antibody epitope (UniProt P69905: isoforms and domain). HBA1 participates in several hemoglobin tetramers, so a positive stain alone cannot identify its partner chain (UniProt P69905: subunit). Check the antibody's documented epitope and specificity before interpreting differences as HBA1-specific expression (standard IHC practice).
How should I adapt this IHC interpretation to multiplex IF?
For multiplex IF, pair the HBA1 channel with an independently validated erythrocyte marker and inspect cell-level overlap, since the expected positive cell type is the erythrocyte (HPA: distinct erythrocyte positivity; standard IF practice). Choose fluorophores and filters after measuring tissue autofluorescence in an unstained section, and include single-stain controls when checking channel bleed-through (standard IF practice). HBA1 has no transmembrane segment, so use only enough permeabilisation to access an intracellular globin epitope without disrupting cell outlines (UniProt P69905: topology and globin domain; standard IF practice). The supplied paraffin IHC caption does not establish an IF fixation or permeabilisation condition; optimise those conditions with IF controls (datasheet A00233-1; standard IF practice).
How can I reduce diffuse brown background around blood-rich regions?
First compare stained tissue with a no-primary control and inspect erythrocyte-rich areas, where true hemoglobin signal may be abundant (HPA: distinct erythrocyte positivity; standard IHC practice). For HRP and DAB detection, include an endogenous peroxidase block and control DAB development time, especially where blood is present (standard IHC practice). The selected image used 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (datasheet A00233-1). If background persists, titrate primary antibody and strengthen washes while checking that the positive control retains a clear cell-associated signal (standard IHC practice).
What is a defensible way to quantify HBA1 IHC staining? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment before scoring, because erythrocytes can occupy different fractions of otherwise similar tissue fields (HPA: distinct erythrocyte positivity; standard IHC practice). Report the percentage of positive erythrocytes or their density per mm² of sampled tissue, and separately record staining intensity or an H-score where cell boundaries permit reliable scoring (standard IHC practice). Normalise erythrocyte counts to the sampled area or an independently measured erythrocyte count, and keep threshold, illumination, counterstain, and DAB development consistent across sections (standard IHC practice). State that this readout measures hemoglobin-antibody staining rather than uniquely quantified HBA1 unless antibody specificity has been established (datasheet A00233-1: anti-Hemoglobin antibody; HPA: multi-gene caution).
Which findings argue that an apparent HBA1-positive cell is artefactual?
A convincing pattern follows identifiable erythrocytes and agrees with the reported selective erythrocyte staining (HPA: tissue IHC profile; UniProt P69905: red blood cell specificity). Treat isolated nuclear staining, staining in unexpected cell types, and diffuse extracellular color as findings that require further checks; UniProt provides no annotated subcellular location to validate a nuclear pattern (UniProt P69905: subcellular location not annotated; standard IHC practice). Check section edges and necrotic areas for uneven reagent exposure or trapped chromogen, and use a no-primary control to assess endogenous enzyme signal (standard IHC practice). Even well-localised positivity does not establish HBA1-specific detection because the available antibody evidence concerns hemoglobin and HPA flags multi-gene reactivity (datasheet A00233-1; HPA: reliability description).
Boster reagents
Best HBA1 / Hemoglobin subunit alpha IHC Antibodies
IHC images show paraffin sections of human placenta and tonsil for A00233-1, and human spleen and mouse embryo for M00233 (catalog IHC captions). Neither has IF imagery (catalog IF image fields).
Real IHC data
Anti-Hemoglobin/HBA1/HBA2 Antibody ®
Cat # A00233-1
Real IHC data
Anti-Hemoglobin subunit alpha HBA1 Rabbit Monoclonal Antibody
Cat # M00233
A00233-1 lists human IHC and shows staining in paraffin sections of human placenta and tonsil (catalog applications, reactivity and IHC captions). M00233 lists IHC with Human, Mouse and Rat reactivity, and shows staining in paraffin sections of human spleen and mouse embryo (catalog applications, reactivity and IHC captions).
Which to pick: For human paraffin-section IHC, choose A00233-1 when its polyclonal Hemoglobin/HBA1/HBA2 target scope suits the experiment; its images show human placenta and tonsil, and the fixative is unreported (catalog title and dilution_raw; A00233-1 IHC captions). For HBA1-focused or cross-species tissue IHC, choose monoclonal M00233, clone EGI-8: it lists Human, Mouse and Rat reactivity, while its paraffin-section images show human spleen and mouse embryo only; the fixative is unreported (catalog title, clone, reactivity and M00233 IHC captions). For IF/ICC, neither SKU has a listed IF/ICC application or IF image, so neither is supported for that use by this catalog evidence (catalog applications and IF image fields).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P69905 (HBA_HUMAN, Hemoglobin subunit alpha).
- Human Protein Atlas. HBA1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HBA1 subcellular location (ICC-IF): Highest expression in HEL: 515.0 nTPM.
- Human Protein Atlas. HBA1 antibody validation summary (2 antibodies).
- Hemoglobin overexpression and splice signature as new features of inflammatory breast cancer?. Journal of advanced research 2021 — PMC7753232.
- Characterization of adult α- and β-globin elevated by hydrogen peroxide in cervical cancer cells that play a cytoprotective role against oxidative insults. PloS one 2013 — PMC3547883.
- Digoxin Attenuates Ocular Hypertension and Protects Trabecular Meshwork Cells via Ferroptosis Inhibition. Investigative ophthalmology & visual science 2026 — PMC12831143.
- PubMed PMID:7448866 — UniProt-cited evidence.
- PubMed PMID:6244294 — UniProt-cited evidence.
- PubMed PMID:6452630 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HBA1 (Hemoglobin subunit alpha). Expected localisation Erythrocyte positivity; intracellular compartment unassigned (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A00233-1). Validated IHC antibody: A00233-1 at 2-5μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bone marrow (Hematopoietic cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human placenta tissue; fixative not specified (datasheet A00233-1); 2. Fixation: Image fixative and duration unreported (datasheet A00233-1); 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: EDTA pH 8.0 (datasheet A00233-1); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A00233-1); 8. Primary antibody: Rabbit anti-HBA1, 2-5μg/ml (datasheet A00233-1); 9. Primary incubation: Overnight at 4 °C (datasheet A00233-1). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); target-specific effects are unknown.; Caveat: HBA2 recognition may confound HBA1 attribution (catalog A00233-1; HPA tissue IHC); Isoform / epitope: 0 isoforms reported; mature chain spans residues 2–142 (UniProt); Regulation: Staining intensity reflects erythrocyte content (UniProt; HPA tissue IHC). Evidence: UniProt P69905, HPA tissue IHC · Enhanced, HPA cell-line RNA · Highest expression in HEL: 515.0 nTPM, Datasheet A00233-1, PMC7753232, PMC3547883. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HBA1/immunohistochemistry-ihc-guide.