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CYB5A / Cytochrome b5 · IHC design guide
Design Immunohistochemistry for CYB5A
Plan chromogenic CYB5A IHC on paraffin sections around the cytoplasmic staining reported in kidney and liver (HPA tissue IHC). Start with the catalog antibody’s IHC dilution range of 1:50–1:200 (datasheet) and assess staining by cell type.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CYB5A 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 IHC; ER membrane is a molecular expectation (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic staining, most abundant in kidney and liver (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Lung+4 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) | |
| Caveat | Adipocytes stain medium in breast but are undetected in adipose tissue (HPA tissue IHC) | |
| Regulation | Expression regulation is not annotated (UniProt) | |
| Isoform / epitope | 3 isoforms; epitope position relative to the transmembrane segment is unknown (UniProt) |
Section 1
Recommended CYB5A IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by a published CYB5A lung-section protocol (PMC4448130 methods).
| Sample | Paraffin-embedded human heart tissue; fixative not specified (datasheet A04464-1) |
| Fixation | Image fixative and duration unreported (datasheet A04464-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane antigen) |
| 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-CYB5A, 1:50-1:200 (datasheet A04464-1) |
| 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 | CYB5A-positive staining in alveolar cells type II of lung (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression most abundant in kidney and liver. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval rule); the published microwave schedule is a separate documented condition (PMC4448130 methods).
Section 2
What Is the Expected CYB5A Staining Pattern?
CYB5A is an endoplasmic reticulum and microsome membrane protein with a transmembrane segment at residues 109–131 (UniProt P00167 topology). In paraffin IHC, expect predominantly cytoplasmic staining, including kidney proximal tubules, lung alveolar type II cells and testicular Leydig cells (HPA tissue IHC). HPA rates tissue staining reliability Enhanced, while reporting only medium consistency between staining and RNA expression (HPA tissue IHC).
| Cytoplasmic signal in kidney proximal tubule cell bodies, with cell boundaries and nuclei discernible. | This matches the reported medium proximal tubule staining and cytoplasmic tissue profile (HPA tissue IHC). A chromogenic section may show a cytoplasmic pattern without resolving individual endoplasmic reticulum membranes (UniProt P00167 subcellular location; standard IHC practice). |
| Strong cytoplasmic staining in lung alveolar type II cells or testicular Leydig cells. | Both cell populations are reported as High and can serve as reference positives (HPA tissue IHC). Compare the expected cell population within each section; staining strength alone does not establish antibody specificity (HPA tissue IHC; standard IHC practice). |
| Predominantly nuclear staining, or a crisp cell surface outline with little cytoplasmic signal. | That compartment pattern conflicts with the cytoplasmic tissue profile and endoplasmic reticulum or microsome membrane annotation (HPA tissue IHC; UniProt P00167 subcellular location). Treat it as suspect and compare with a known-positive section and detection controls (standard IHC practice). |
| Strong signal in a cell population reported as Not detected, such as appendix glandular cells. | This differs from that HPA observation and warrants a cross-reactivity or endogenous detection activity check (HPA tissue IHC; standard IHC practice). Not detected in an HPA sample is a comparison point, not proof that every specimen must be negative (HPA tissue IHC). |
| Uniform haze across cells and surrounding tissue, or no signal in a known-positive section. | Haze obscures the cell-specific cytoplasmic pattern; inspect background controls and detection steps (HPA tissue IHC; standard IHC practice). An absent reference-positive signal leaves the run uninterpretable until the assay and tissue quality are checked (standard IHC practice). |
💡Expected CYB5A appearanceCall a result positive when cytoplasmic signal follows the reported cell pattern—High in alveolar type II or Leydig cells, or Medium in kidney proximal tubules; predominant nuclear staining is suspect (HPA tissue IHC; UniProt P00167 subcellular location).
| Membrane topology and epitope location | CYB5A has one transmembrane segment at 109–131 and a heme-binding domain at 9–85 (UniProt P00167 topology; domains). The antibody epitope is unspecified, so these annotations cannot predict its retrieval requirement (UniProt P00167 topology; standard IHC practice). |
| Isoforms and processing | 3 isoforms are listed, with a chain annotated at residues 2–134; no signal peptide or propeptide is listed (UniProt P00167 isoforms; processing). The supplied record does not show which isoforms the IHC-validated antibody detects. |
| Reference tissue selection | HPA reports High staining in lung alveolar type II and testicular Leydig cells, and Medium staining in kidney proximal tubules (HPA tissue IHC). Its broader profile says cytoplasmic expression is most abundant in kidney and liver without specifying a liver cell type (HPA tissue IHC). |
| Strength of the staining evidence | HPA labels tissue IHC reliability Enhanced but describes medium consistency with RNA expression (HPA tissue IHC). Its listed antibodies have Enhanced IHC status; this supports using the reported pattern as a reference while retaining sample and assay controls (HPA antibodies; standard IHC practice). |
| Detection and background | Endogenous enzyme activity and nonspecific reagent binding can create chromogenic background (standard IHC practice). Check the detection controls and blocking conditions when diffuse or unexpected staining appears; these are general IHC checks, not documented CYB5A-specific effects (standard IHC practice). |
| IF/ICC Q: Should its pattern match tissue IHC exactly? | A: HPA ICC-IF reports vesicles and cytosol in Hep-G2, RT-4 and U2OS, while tissue IHC is described as cytoplasmic (HPA subcellular; HPA tissue IHC). Use the separate IF/ICC guide for that application; the supplied IHC evidence does not define an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No stain in a lung or testis reference-positive cell population. | The section may have failed to yield interpretable signal; the payload does not identify a CYB5A-specific fixation or retrieval effect (HPA tissue IHC; standard IHC practice). | Confirm the expected cells are present, inspect tissue quality, and check the IHC-validated antibody and detection steps against the run controls (standard IHC practice). |
| Signal appears mainly in nuclei. | This disagrees with the cytoplasmic tissue pattern and membrane-associated annotation (HPA tissue IHC; UniProt P00167 subcellular location). | Review counterstain and localization, then compare the result with a known-positive section and an appropriate detection control (standard IHC practice). |
| Chromogen appears broadly in cells expected to show little or no staining. | Cross-reactivity or endogenous detection activity is possible (standard IHC practice); HPA lists appendix glandular cells as Not detected (HPA tissue IHC). | Check a detection-only control and the relevant endogenous activity blocking step; judge the result against cell-level staining, not tissue name alone (standard IHC practice). |
| Diffuse haze makes cytoplasmic staining hard to score. | Nonspecific reagent binding or excess detection background may obscure the reported cytoplasmic pattern (standard IHC practice; HPA tissue IHC). | Inspect background controls and review blocking, wash and detection conditions before scoring the section (standard IHC practice). |
| Kidney proximal tubules stain less strongly than lung reference cells. | That comparison can fit HPA's Medium kidney proximal tubule and High lung alveolar type II observations (HPA tissue IHC). | Score the expected cell population and localization within each tissue; do not require equal intensity across those references (HPA tissue IHC; standard IHC practice). |
| Adipocytes stain in breast but appear unstained in adipose tissue. | HPA records Medium breast adipocytes and Not detected adipose tissue adipocytes, so its observations differ by sampled tissue (HPA tissue IHC). | Record the tissue context and controls before calling either result an assay failure; avoid treating one adipocyte observation as a universal rule (HPA tissue IHC; standard IHC practice). |
Experiment evidence
IHC & IF images showing CYB5A staining
A04464-1 · IHCImmunohistochemistry (IHC) analyzes of Cytochrome b5 (G82) pAb in paraffin-embedded human heart tissue.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166347-CYB5A/tissue/cerebral+cortex#imid_17099779
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166347-CYB5A/tissue/colon#imid_17099705
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166347-CYB5A/tissue/liver#imid_17099690
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000166347-CYB5A/tissue/kidney#imid_17099699
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA047548; green = CYB5A, blue = nucleus, red = microtubules.
HPA · ICC-IF Hep-G2Human Protein Atlas immunofluorescence, Hep-G2, antibody HPA047548; green = CYB5A, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CYB5A IHC & IF
🧪Run lung first: alveolar type II cells should stain (HPA: High in alveolar cells type II). Use appendix glandular cells as the negative tissue (HPA: Not detected); adjacent lung cells without staining can provide a within-slide background reference, but are not a validated biological negative.
Positive control tissue: Lung (Alveolar cells type II, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CYB5A in Hep-G2, RT-4, U2OS, with annotated localisation: Vesicles (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; host-species-matched nonimmune IgG for the catalog polyclonal antibody (caption: pAb); and CYB5A knockout tissue or a validated peptide-block control (standard IHC practice). For chromogenic lung IHC, quench endogenous peroxidase and check background on the no-primary slide (standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A04464-1 paraffin-section caption does not state the fixative (caption: fixative not stated). Retrieval dependency is unreported, so optimize antigen retrieval empirically (standard IHC practice); the supplied evidence does not establish whether frozen sections or IF are easier. Lung sections may show endogenous peroxidase background, which the no-primary control helps assess (standard IHC practice).
HPA tissue IHC evidence for CYB5A
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 |
|---|---|---|---|---|
| Lung | Alveolar cells type II | High | Protein (IHC) | HPA → |
| Testis | Leydig cells | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Adipocytes | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| 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 → |
Section 3
Advanced CYB5A IHC Tips
Troubleshoot CYB5A staining in paraffin sections by checking retrieval, cellular pattern, controls, and scoring against the supplied tissue evidence.
How should I optimize retrieval when CYB5A staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 min (page retrieval setting). If staining is weak, compare a longer incubation in the same buffer on matched sections before trying a different buffer or pH; keep the detection and imaging conditions fixed so the comparison is interpretable (standard IHC practice). Include a kidney section and assess proximal tubule cell bodies as a reference pattern (HPA: medium staining in kidney proximal tubules). Excess retrieval can damage morphology or raise diffuse background, so judge the result by preserved tissue structure as well as signal (standard IHC practice).
Could fixation explain weak staining in my paraffin sections?
CYB5A specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence; the catalog image identifies paraffin embedded human heart tissue but does not state its fixative (selected A04464-1 tissue IHC caption). Record each specimen’s fixative, fixation interval, and processing history before comparing staining across blocks (standard IHC practice). For troubleshooting, stain matched sections together using the page’s citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval setting). If signal varies between blocks, examine morphology and processing records before attributing the difference to CYB5A abundance (standard IHC practice).
Where should convincing CYB5A staining appear in tissue?
Expect a predominantly cytoplasmic pattern in appropriate cells, with a possible fine membranous appearance consistent with CYB5A at the endoplasmic reticulum and microsome membranes (HPA: cytoplasmic tissue expression; UniProt P00167: subcellular location). The protein has a transmembrane segment at residues 109–131, so a sharp nuclear only pattern warrants scrutiny (UniProt P00167: topology; standard IHC interpretation). Use kidney proximal tubule cell bodies as a reference, where HPA reports medium staining (HPA: kidney proximal tubules). Compare cell distribution, morphology, and a no primary control before accepting an unfamiliar compartment pattern as specific (standard IHC practice).
Can this antibody distinguish CYB5A isoforms or epitope accessibility?
The record lists 3 CYB5A isoforms, but the supplied antibody information does not identify its epitope or establish isoform selectivity (UniProt P00167: isoforms; supplied A04464-1 caption). CYB5A contains a heme binding domain at residues 9–85 and a transmembrane segment at 109–131; those coordinates alone cannot establish which forms this antibody recognizes (UniProt P00167: domains and topology). If isoform discrimination matters, obtain the immunogen sequence and compare it with each isoform before interpreting staining (standard antibody validation practice). Likewise, altered staining after retrieval cannot by itself identify the bound epitope or prove that a specific modification masked it (standard IHC interpretation).
How can I cross check the chromogenic pattern by immunofluorescence?
Treat IF as a separate assay and first confirm that the antibody works under the chosen fixation and permeabilisation conditions; the paraffin tissue caption supplies no IF fixation evidence (selected A04464-1 tissue IHC caption). Multiplex CYB5A with a validated marker for the expected cell type, such as a proximal tubule marker when examining kidney, and compare cell level overlap rather than treating shared color as proof of specificity (HPA: kidney proximal tubules; standard IF practice). Choose fluorophores after checking unstained tissue for autofluorescence, and include single stain controls for bleed through (standard IF practice). Because CYB5A is membrane associated with a cytoplasmic domain, adjust permeabilisation to expose the antibody’s mapped epitope; its epitope is not supplied here (UniProt P00167: topology; supplied A04464-1 caption).
What should I check when DAB background obscures CYB5A?
First inspect a no primary control and the tissue edges to distinguish detection background from cell associated staining (standard chromogenic IHC practice). Use a peroxidase block before DAB detection, then check whether residual color persists without primary antibody; these are general chromogenic workflow steps, not evidence that CYB5A itself causes background (standard IHC practice). Compare the catalog antibody at a small dilution series while holding citrate pH 6.0 retrieval, 95–98 °C, and 20 min constant (page retrieval setting; standard IHC optimization). Accept a condition only when background falls without losing the expected cytoplasmic pattern in kidney proximal tubule cell bodies (HPA: cytoplasmic profile and kidney proximal tubules).
How should I score CYB5A across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the scored cell population and compartment before analysis; CYB5A has a cytoplasmic tissue profile, and kidney proximal tubule cell bodies provide one documented population (HPA: tissue profile and kidney proximal tubules). Report an H score from intensity and percentage of positive cells, or report percentage positive cells within a specified region; apply the same thresholds across sections (standard IHC quantification practice). Normalize counts to the number of eligible cells or sampled tissue area in mm², and keep section thickness and imaging settings consistent (standard IHC practice). Exclude folds, necrosis, and section edges by a predefined rule, then retain representative images and control results for review (standard IHC practice).
How do I distinguish real CYB5A signal from artefact?
A credible pattern is cell associated and predominantly cytoplasmic, consistent with CYB5A’s endoplasmic reticulum and microsome membrane location and HPA’s cytoplasmic tissue profile (UniProt P00167: subcellular location; HPA: tissue profile). Check whether stained cells fit the specimen: HPA reports medium signal in kidney proximal tubule cell bodies and high signal in lung alveolar type II cells (HPA: kidney and lung tissue IHC). Nuclear only staining, staining confined to cut edges or necrosis, and color in a no primary control call for investigation of compartment mismatch, section artefact, or endogenous enzyme activity (standard IHC interpretation). Treat a matching pattern as supportive rather than conclusive because HPA rates tissue staining “Enhanced” with medium consistency against RNA expression (HPA: reliability description).
Boster reagents
Best CYB5A / Cytochrome b5 IHC Antibodies
A04464-1 is listed for CYB5A IHC and IF in human, mouse, and rat (catalog: applications/reactivity). Its IHC image shows a paraffin-embedded human heart section (image caption).
Real IHC data
Anti-Cytochrome b5 (G82) CYB5A Antibody
Cat # A04464-1
A04464-1 will render with its own IHC image of paraffin-embedded human heart tissue (image caption). It lists IHC and IF applications and human, mouse, and rat reactivity (catalog: applications/reactivity); no IF image is supplied (catalog: IF image alts).
Which to pick: For tissue IHC, choose A04464-1: its image documents a paraffin-embedded human heart section, but does not report the fixative (image caption). For IF/ICC, A04464-1 lists IF, while ICC validation and an IF image are not supplied; it is identified as a rabbit polyclonal antibody (catalog: applications/host/IF image alts; image caption: pAb). For cross-species work, A04464-1 lists human, mouse, and rat reactivity, although its IHC image documents human tissue alone (catalog: reactivity; image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P00167 (CYB5_HUMAN, Cytochrome b5).
- Human Protein Atlas. CYB5A tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. CYB5A subcellular location (ICC-IF): Localized to vesicles and cytosol..
- Human Protein Atlas. CYB5A antibody validation summary (3 antibodies).
- 3βHSD and CYB5A double positive adrenocortical cells during adrenal development/aging. Endocrine research 2015 — PMC4242803.
- The proteome of Hypobaric Induced Hypoxic Lung: Insights from Temporal Proteomic Profiling for Biomarker Discovery. Scientific reports 2015 — PMC4448130.
- Adrenal tropism of SARS-CoV-2 and adrenal findings in a post-mortem case series of patients with severe fatal COVID-19. Nature communications 2022 — PMC8948269.
- Morphologic and Molecular Characterization of Adrenals and Adrenal Rest Affected by Congenital Adrenal Hyperplasia. Frontiers in endocrinology 2021 — PMC8488225.
- PubMed PMID:3178851 — UniProt-cited evidence.
- PubMed PMID:1712589 — UniProt-cited evidence.
- PubMed PMID:7733981 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CYB5A (Cytochrome b5). Expected localisation Cytoplasmic IHC; ER membrane is a molecular expectation (HPA tissue IHC; UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A04464-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Lung (Alveolar cells type II); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human heart tissue; fixative not specified (datasheet A04464-1); 2. Fixation: Image fixative and duration unreported (datasheet A04464-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 20 min at 95–98 °C…; 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-CYB5A, 1:50-1:200 (datasheet A04464-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Adipocytes stain medium in breast but are undetected in adipose tissue (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope position relative to the transmembrane segment is unknown (UniProt); Regulation: Expression regulation is not annotated (UniProt). Evidence: UniProt P00167, HPA tissue IHC · Enhanced, HPA ICC-IF · vesicles and cytosol., Datasheet A04464-1, PMC4448130. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYB5A/immunohistochemistry-ihc-guide.