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CYB5A / Cytochrome b5 · IHC design guide
Design Immunohistochemistry for CYB5A
Plan CYB5A chromogenic IHC around cytoplasmic staining most abundant in kidney and liver (HPA tissue IHC). Use the IHC-validated antibody at a starting dilution within 1:50–1:200, then optimize locally (datasheet).
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 tissue staining (HPA tissue IHC) | |
| 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 | Antibody staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Kidney- and liver-enhanced RNA expression (HPA tissue RNA) | |
| Isoform / epitope | 3 isoforms; epitope coverage is unknown (UniProt) |
Section 1
Recommended CYB5A IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with three published CYB5A IHC workflows (PMC4448130; PMC8948269; PMC8488225).
| 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); adjust against the published microwave method (PMC4448130).
Section 2
What Is the Expected CYB5A Staining Pattern?
CYB5A is associated with the endoplasmic reticulum and microsome membranes and is also annotated in the cytoplasm; its transmembrane segment spans residues 109–131 (UniProt P00167 topology). Expect cytoplasmic IHC staining in selected cells, especially kidney and liver overall, with high staining reported in lung alveolar type II cells and testicular Leydig cells (HPA: tissue IHC). HPA rates the tissue profile Enhanced, with medium agreement between staining and RNA data (HPA: reliability).
| Cytoplasmic staining in alveolar type II or Leydig cells, with nuclei comparatively clear. | This fits the reported High staining in those cell populations (HPA: lung and testis IHC). Score the named cells separately from neighboring cells: staining intensity across an entire tissue section can hide whether the expected population carries the signal (standard IHC practice). |
| Cytoplasmic staining in kidney proximal tubule cell bodies, with a less intense result than the high-staining examples. | A Medium proximal tubule result matches the listed kidney observation (HPA: kidney IHC). The broader profile says expression is most abundant in kidney and liver; it does not assign every kidney cell or a specific liver cell an intensity (HPA: tissue IHC profile). |
| Predominantly nuclear staining, or a crisp cell-surface outline without convincing cytoplasmic signal. | Treat that compartment pattern as suspect: the reported tissue pattern is cytoplasmic (HPA: tissue IHC), while UniProt places CYB5A at endoplasmic reticulum and microsome membranes and in cytoplasm (UniProt P00167 subcellular location). Review controls and detection conditions before assigning biological meaning (standard IHC practice). |
| Strong staining in a cell population listed as not detected, such as appendix glandular cells. | This conflicts with that specific HPA observation (HPA: appendix glandular cells, Not detected). Consider cross-reactivity or endogenous detection activity and compare a known-positive section processed alongside it (standard IHC practice). HPA entries describe sampled cell populations; they are not a universal negative-cell atlas (HPA: tissue IHC). |
| Diffuse color across tissue and empty spaces, or no staining in a known-positive section. | Color outside cells is background rather than a localizable CYB5A pattern (standard IHC practice). A blank high-staining reference, such as alveolar type II or Leydig cells, makes a technical failure plausible; it cannot establish CYB5A absence in an experimental specimen (HPA: lung and testis IHC; standard IHC practice). |
💡Expected CYB5A appearanceCall a result positive when identifiable alveolar type II or Leyig cells show High cytoplasmic staining, or kidney proximal tubule cell bodies show Medium cytoplasmic staining (HPA: lung, testis and kidney IHC); nuclear-only or widespread extracellular color is suspect (HPA: tissue IHC; standard IHC practice).
| Membrane location and topology | CYB5A has one transmembrane segment at residues 109–131 and an annotated endoplasmic reticulum and microsome location (UniProt P00167 topology and subcellular location). Interpret chromogenic signal within the cytoplasmic compartment; these annotations alone do not establish an exact microscopic texture (HPA: cytoplasmic tissue IHC). |
| Reference tissue and cell selection | Lung alveolar type II and testicular Leydig cells are listed as High; kidney proximal tubule cell bodies are Medium (HPA: tissue IHC). Use the cell identity and its listed level together when judging a control; a medium result need not match a high result in intensity (standard IHC practice). |
| Strength of the tissue evidence | The tissue profile is Enhanced, with medium consistency between antibody staining and RNA expression (HPA: reliability). Three listed antibodies have Enhanced IHC status (HPA: antibody validation). These ratings support the reported pattern but do not guarantee that every specimen or assay condition reproduces it (standard IHC practice). |
| Context within a named cell type | Breast adipocytes are listed as Medium, whereas adipocytes in adipose tissue are listed as Not detected (HPA: breast and adipose tissue IHC). Preserve the tissue context when choosing controls or assessing an apparent exception; the shared cell label alone does not predict staining across sites (HPA: tissue IHC). |
| Isoforms and antigen retrieval | UniProt lists 3 isoforms, but the supplied record does not map antibody epitopes to them (UniProt P00167 isoforms). No target-specific retrieval or fixation effect is supplied (UniProt P00167; HPA: tissue IHC). Any retrieval comparison is assay optimization, not evidence that CYB5A requires a particular condition (standard IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in the experimental section or its known-positive control. | A blank positive reference suggests an assay or specimen-processing problem; it does not establish absent CYB5A (HPA: lung alveolar type II and testis Leydig cells, High; standard IHC practice). | Check section integrity, antibody and detection steps, then compare retrieval conditions using matched sections (standard IHC practice). Reassess the experimental section only after the reference cells show their expected cytoplasmic pattern (HPA: tissue IHC). |
| A reference stains, but the signal is mainly nuclear. | The compartment conflicts with HPA cytoplasmic tissue staining and UniProt membrane and cytoplasm annotations (HPA: tissue IHC; UniProt P00167 subcellular location). This raises an assay-specificity concern rather than proving nuclear CYB5A. | Compare the same cell population with another listed Enhanced IHC antibody if available, and check control and detection behavior (HPA: antibody validation; standard IHC practice). Record the discordant compartment instead of scoring it as expected staining. |
| Broad brown haze obscures cell boundaries. | Diffuse noncellular color prevents a confident compartment or cell-type call (standard IHC practice). It cannot be matched to the reported cytoplasmic pattern in named cells (HPA: tissue IHC). | Inspect a detection-only control and optimize blocking, washing, antibody concentration and detection development on matched sections (standard IHC practice). Re-score only when the expected cells can be distinguished from their surroundings. |
| An HPA-listed negative cell population stains strongly. | For example, staining in appendix glandular cells conflicts with their Not detected entry (HPA: appendix IHC). Cross-reactivity or endogenous detection activity is possible; a single discrepant section cannot distinguish them (standard IHC practice). | Compare a listed high-staining cell population in the same run and use a detection-only control to probe reagent-derived color (HPA: lung and testis IHC; standard IHC practice). Confirm the cell identity before calling the result discrepant. |
| The kidney section is weaker than a lung or testis reference. | That difference can fit the listed levels: kidney proximal tubule cell bodies are Medium, while alveolar type II and Leydig cells are High (HPA: tissue IHC). | Judge each reference against its own reported cell population and level, with matched staining conditions (HPA: tissue IHC; standard IHC practice). Investigate weak kidney staining only if its expected proximal tubule pattern is absent or unreadable. |
| What if an IF/ICC image looks vesicular or cytosolic? | HPA reports vesicles and cytosol as enhanced IF/ICC locations, while its tissue IHC description is cytoplasmic (HPA: subcellular ICC-IF; HPA: tissue IHC). The image types resolve and report location differently (standard microscopy practice). | Use the IF/ICC observation as a location cross-check, and judge the paraffin IHC section by cell identity and cytoplasmic staining (HPA: subcellular ICC-IF; HPA: tissue IHC). Consult the separate IF/ICC guide for that assay. |
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 in appendix glandular cells); on the lung slide, use nearby cells lacking specific staining as an internal background reference, without assuming all other lung cells are CYB5A 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; an isotype control matched to the primary antibody’s host species, clonality and concentration; and CYB5A knockout material processed alongside the positive sample as a biological negative (standard IHC practice). For chromogenic lung IHC, block endogenous peroxidase and inspect pigment-associated background before scoring (standard IHC practice).
⚠️Feasibility: No target-specific fixation window or fixation effect is reported in the supplied evidence, and the paraffin-embedded heart caption for A04464-1 does not state a fixative (selected tissue-IHC caption). Retrieval dependence is unreported, so optimize antigen retrieval for the catalog antibody’s IHC-P protocol (standard IHC practice). The evidence does not establish that frozen sections or IF are easier; in lung, pigment and endogenous peroxidase can complicate chromogenic scoring (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 chromogenic IHC in paraffin sections using matched controls, compartment-aware scoring, and the antibody’s documented tissue evidence.
How should I retrieve CYB5A when staining is weak in paraffin sections?
Start with citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min (page retrieval rule). Cool the slides in buffer, then compare staining in kidney proximal tubules with a no-primary control (standard IHC practice; HPA: medium in kidney proximal tubules). Keep section thickness, antibody dilution, and chromogen development constant while assessing retrieval (standard IHC practice). If staining remains weak, test a second retrieval condition on adjacent sections and inspect both signal and tissue morphology before adopting it (standard IHC practice). The catalog image documents CYB5A staining in paraffin-embedded human heart, but its caption does not specify a retrieval method (catalog caption: A04464-1).
Could fixation explain weak or patchy CYB5A staining?
CYB5A-specific sensitivity to fixation is unknown from the supplied evidence, so treat fixation as an experimental variable rather than an established cause (supplied evidence: no target-specific fixation comparison). The catalog image identifies paraffin-embedded human heart tissue but does not state its fixative (catalog caption: A04464-1). Record the fixative, fixation duration, tissue thickness, and processing history for each specimen, then compare matched sections under the same retrieval and detection conditions (standard IHC practice). If staining varies across a block, assess morphology and compare internal cell populations before changing antibody concentration (standard IHC practice). Do not infer fixation sensitivity from CYB5A topology, acetylation, or HPA tissue patterns (UniProt P00167 topology and modified residues; HPA: tissue IHC profile).
Where should convincing CYB5A signal appear in chromogenic IHC?
Expect predominantly cytoplasmic staining associated with membrane-rich compartments, since CYB5A is assigned to the endoplasmic reticulum and microsome membranes and has a transmembrane segment at residues 109–131 (UniProt P00167 localisation and topology). HPA describes cytoplasmic tissue expression, most abundant in kidney and liver (HPA: tissue IHC profile). Score signal within identifiable cells and compare its distribution with adjacent no-primary and positive-control sections (standard IHC practice). Diffuse nuclear-only staining would conflict with the supplied CYB5A localisation evidence and merits a specificity check (UniProt P00167 localisation; standard IHC practice). HPA’s vesicle and cytosol assignments come from subcellular imaging and need not appear as resolvable structures in chromogenic tissue sections (HPA: subcellular profile; standard IHC practice).
How should isoforms and epitope position affect antibody validation?
CYB5A has 3 listed isoforms, and the supplied record does not specify which sequences the catalog antibody recognizes (UniProt P00167 isoforms; supplied evidence: no epitope map). Check the antibody’s documented immunogen or epitope against each isoform before interpreting absent staining as absent total CYB5A (standard IHC practice). The heme-binding domain spans residues 9–85, whereas the transmembrane segment spans 109–131, making epitope position relevant to tissue processing and accessibility (UniProt P00167 domains and topology; standard IHC practice). Acetylation is listed at residues 2, 10, and 19, but no supplied evidence shows that these modifications alter this antibody’s IHC signal (UniProt P00167 modified residues; supplied evidence: no antibody-specific comparison). Compare antibody staining with an independent specificity control when isoform coverage matters (standard IHC practice).
How can I compare CYB5A immunofluorescence with tissue IHC?
Treat IF as a separate assay: HPA reports vesicle and cytosol localisation in cell imaging, while tissue IHC reports cytoplasmic staining (HPA: subcellular profile; HPA: tissue IHC profile). Multiplex CYB5A with a validated marker for the expected cell population, such as kidney proximal tubules, and include single-stain controls to check channel bleed-through (HPA: medium in kidney proximal tubules; standard IF practice). Select a fluorophore in a channel with low measured tissue autofluorescence, and set exposure using unstained and no-primary sections (standard IF practice). Choose permeabilisation after establishing whether the antibody epitope is exposed to the cytosol or another membrane side; its orientation is not supplied here (UniProt P00167 topology; supplied evidence: no epitope orientation). Do not transfer fixation settings from the paraffin heart caption to IF (catalog caption: A04464-1).
What should I check when CYB5A chromogen appears broadly brown?
First compare the stained section with a no-primary control and inspect whether brown signal follows cells or accumulates at folds and cut edges (standard IHC practice). Block endogenous peroxidase before peroxidase-based DAB detection, and verify that the blocking step reduces signal in the no-primary section (standard IHC practice). Titrate the primary antibody and shorten chromogen development if background rises while the positive-control pattern remains visible (standard IHC practice). Assess nonspecific secondary-reagent binding with an appropriate control and keep wash conditions consistent across slides (standard IHC practice). Because CYB5A is membrane-associated and HPA reports cytoplasmic tissue staining, widespread extracellular deposit or uniform nuclear color needs further investigation (UniProt P00167 localisation; HPA: tissue IHC profile).
How should I quantify CYB5A across specimens with different cell mixtures? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and tissue region before scoring, using morphology or a matched cell-type marker where needed (standard IHC practice). Record either an H-score combining intensity and percentage of positive cells, or the percentage positive at a prespecified threshold; keep the same method across sections (standard IHC practice). Normalise counts to the number of evaluable cells in the chosen population, or report positive-cell density per mm² of evaluable tissue (standard IHC practice). Exclude folds, necrosis, and section edges using the same criteria for every specimen (standard IHC practice). Cell composition matters because HPA reports medium staining in kidney proximal tubules and high staining in lung alveolar type II cells and testis Leydig cells (HPA: tissue IHC profile).
How can I distinguish true CYB5A staining from artefact?
Look for cellular cytoplasmic signal compatible with CYB5A’s endoplasmic reticulum and microsome association, then compare it with a no-primary section (UniProt P00167 localisation; standard IHC practice). Use the reported high staining in lung alveolar type II cells or testis Leydig cells as context when suitable control tissue is available (HPA: tissue IHC profile). Treat nuclear-only staining, edge-restricted color, and deposits in necrotic areas as reasons to investigate the result before calling cells positive (UniProt P00167 localisation; standard IHC practice). Check endogenous peroxidase with a no-primary control when interpreting DAB signal (standard IHC practice). Interpret negative cells cautiously: HPA rates tissue staining reliability as Enhanced but describes only medium consistency with RNA expression data (HPA: tissue IHC reliability).
Boster reagents
Best CYB5A / Cytochrome b5 IHC Antibodies
A04464-1 has an IHC image from paraffin-embedded human heart (IHC image caption) and is listed for IF and for human, mouse, and rat reactivity (catalog: applications and reactivity).
Real IHC data
Anti-Cytochrome b5 (G82) CYB5A Antibody
Cat # A04464-1
A04464-1 has an IHC image from paraffin-embedded human heart (A04464-1 IHC image caption). It is also listed for IF and human, mouse, and rat reactivity; no IF image is supplied (catalog: applications, reactivity, IF image alts).
Which to pick: For tissue IHC, choose A04464-1 for paraffin sections; its own image shows human heart, and the fixative is unreported (A04464-1 IHC image caption). For IF, A04464-1 lists a 1:50–1:200 dilution, but ICC is not listed and no IF image is supplied (catalog: applications, IF dilution, IF image alts). For cross-species work, A04464-1 is a rabbit antibody listed as reactive with human, mouse, and rat; the supplied IHC image documents human tissue only (catalog: host and reactivity; A04464-1 IHC 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 tissue staining (HPA tissue IHC). 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: Antibody staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: 3 isoforms; epitope coverage is unknown (UniProt); Regulation: Kidney- and liver-enhanced RNA expression (HPA tissue RNA). Evidence: UniProt P00167, HPA tissue IHC · Enhanced, HPA ICC-IF · vesicles and cytosol., Datasheet A04464-1, PMC4448130, PMC8948269. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CYB5A/immunohistochemistry-ihc-guide.