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COL6A3 / Collagen alpha-3(VI) chain · IHC design guide
Design Immunohistochemistry for COL6A3
Plan COL6A3 paraffin IHC around extracellular matrix positivity and cytoplasmic staining in a few tissues (HPA tissue IHC). Use ovarian stroma as a positive tissue control (HPA tissue IHC), start the catalog antibody at 1:100–1:300 (datasheet), and score matrix and cellular staining separately.
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
COL6A3 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 | Extracellular matrix; some cytoplasmic tissue staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in a few tissues; extracellular matrix positive (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| 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 formalin fixation consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Secreted variants can uncouple tissue RNA and protein staining (HPA tissue IHC) | |
| Regulation | Tissue-enhanced RNA in smooth muscle (HPA tissue RNA) | |
| Isoform / epitope | Five isoforms; mature chain starts at residue 26 (UniProt) |
Section 1
Recommended COL6A3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by four published COL6A3 IHC protocols (PMC3163121; PMC7061042; PMC13424786; PMC4745773).
| Sample | Paraffin-embedded human breast carcinoma tissue; fixative not specified (datasheet A02844) |
| Fixation | Image fixative and duration unreported (datasheet A02844); 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-COL6A3, 1:100 - 1:300 (datasheet A02844) |
| 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 | COL6A3-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: Medium). HPA tissue profile: Cytoplasmic expression in a few tissues with positivity in extracellular matrix. 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). Use each article’s stated retrieval conditions when reproducing its protocol.
Section 2
What Is the Expected COL6A3 Staining Pattern?
COL6A3 is a secreted extracellular matrix protein with no transmembrane segment (UniProt P12111 topology). In paraffin IHC, expect extracellular matrix positivity and cytoplasmic staining in a few tissues, including medium staining in ovarian stromal cells and bone marrow hematopoietic cells (HPA tissue IHC). HPA rates the tissue profile Supported but reports low consistency between staining and RNA expression; a secreted variant can separate the protein signal from its site of production (HPA tissue IHC).
| Extracellular matrix staining accompanies cytoplasmic staining in some cells. | This fits the reported IHC profile (HPA tissue IHC) and the protein’s secreted, extracellular matrix location (UniProt P12111). Assess the cell staining and surrounding matrix separately: secreted protein can appear beyond the cells that produced it (HPA tissue IHC). |
| Ovarian stromal cells or bone marrow hematopoietic cells show moderate signal. | These are the supplied medium-positive tissue and cell examples (HPA tissue IHC). Use the named cell population when judging a positive section; positivity in another compartment of the same tissue does not establish the expected pattern (HPA tissue IHC). |
| Staining is predominantly nuclear, with little cytoplasmic or matrix signal. | A nuclear-dominant pattern does not match the reported tissue IHC profile (HPA tissue IHC) or secreted extracellular matrix location (UniProt P12111). Treat it as a suspect signal and check controls and detection conditions before assigning it to COL6A3. |
| Strong staining appears in an HPA-listed negative cell population, such as adipocytes. | HPA reports adipocytes in adipose tissue as not detected (HPA tissue IHC). Check for nonspecific binding or endogenous detection activity using appropriate controls (general IHC practice). This observation alone cannot identify which source caused the signal. |
| A known-positive cell population lacks signal, or brown color is diffuse across the section. | Absent signal in ovarian stromal or bone marrow hematopoietic cells conflicts with their reported medium staining (HPA tissue IHC); diffuse color without a defined cell or matrix pattern is hard to interpret. Review the positive and negative controls, retrieval, antibody concentration, and detection background (general IHC practice). |
💡Expected COL6A3 appearanceCall a section positive when staining is interpretable in extracellular matrix and/or the reported cytoplasmic cell pattern, including medium signal in ovarian stromal or bone marrow hematopoietic cells (HPA tissue IHC); isolated nuclear staining or diffuse, unstructured color is suspect against that reference pattern (HPA tissue IHC).
| Secreted protein and tissue location | COL6A3 is secreted and associated with extracellular matrix (UniProt P12111). HPA cautions that at least one variant is secreted, so the stained location and the cells expressing its RNA can differ (HPA tissue IHC). Score deposited matrix without assuming the adjacent cell synthesized it. |
| Strength of the tissue reference | The HPA tissue IHC profile is Supported, while HPA also reports low consistency between antibody staining and RNA expression (HPA tissue IHC). Use the named positive cell populations as practical references; avoid treating RNA abundance alone as an IHC pass or fail criterion. |
| Cell population used as a negative reference | HPA reports no detected staining in adipocytes of adipose tissue and several other specified cell populations (HPA tissue IHC). A negative call applies to the listed cells, not automatically to every structure in that organ (HPA tissue IHC). |
| Protein variants and maturation | UniProt lists five isoforms and a signal peptide at residues 1–25 (UniProt P12111). Those facts alone do not establish which isoforms a particular antibody detects or whether processing changes its IHC signal. Confirm the catalog antibody’s epitope information before making an isoform-specific interpretation. |
| Antibody validation scope | The listed HPA IHC antibody, HPA010080, is Supported for IHC; HPA010007 is Approved for ICC and has no listed IHC status (HPA antibodies). An ICC result does not independently validate a paraffin IHC pattern. Assess the IHC-validated antibody against the tissue references and controls. |
| IF/ICC Q: Why might cells show an intracellular pattern? | A: HPA reports mainly endoplasmic reticulum and additional vesicle localization in ICC-IF (HPA subcellular). This can describe intracellular protein before secretion (UniProt P12111); it does not replace the extracellular matrix and cytoplasmic tissue IHC reference (HPA tissue IHC). |
| Situation | Likely cause | Next action |
|---|---|---|
| Ovarian stromal cells or bone marrow hematopoietic cells are unstained. | These populations have medium staining in the HPA reference (HPA tissue IHC); the failed signal has no established target-specific cause from the supplied evidence. | Check that the intended cells are present and review a positive control, retrieval conditions, antibody dilution, and detection reagents (general IHC practice). Interpret a negative test section only after the control works. |
| The whole section has diffuse brown background. | Diffuse color lacks the defined cell or matrix distribution in the reported profile (HPA tissue IHC). Excess reagent, incomplete blocking, or endogenous detection activity are possible general IHC causes. | Compare the test slide with a no-primary control; review blocking, washes, detection chemistry, and antibody concentration (general IHC practice). Do not score diffuse background as extracellular matrix positivity. |
| Nuclei are the main stained structures. | Nuclear-dominant staining is inconsistent with the tissue profile (HPA tissue IHC) and the secreted extracellular matrix annotation (UniProt P12111); its mechanism is undetermined. | Check the no-primary control and the expected positive tissue pattern, then review antibody specificity and detection conditions (general IHC practice). Avoid a COL6A3-positive call from nuclear staining alone. |
| Adipocytes in adipose tissue stain strongly. | HPA reports adipocytes in adipose tissue as not detected (HPA tissue IHC). Nonspecific binding or endogenous detection activity may explain an unexpected signal (general IHC practice). | Confirm the stained cells are adipocytes, inspect the no-primary control, and review blocking and detection chemistry (general IHC practice). Do not extend the adipocyte reference to every other structure in adipose tissue. |
| Matrix stains, but nearby cells have little cytoplasmic signal. | Secreted COL6A3 can reside in extracellular matrix (UniProt P12111), and HPA cautions that protein and RNA locations can differ for a secreted variant (HPA tissue IHC). | Document matrix and cellular staining separately. Compare morphology and a reference positive population before calling the section negative; absence from adjacent cells alone does not invalidate matrix staining (HPA tissue IHC). |
| An ICC-IF image appears vesicular while the IHC slide highlights matrix. | HPA reports endoplasmic reticulum and vesicle localization in ICC-IF (HPA subcellular), while tissue IHC includes extracellular matrix positivity (HPA tissue IHC). | Judge the paraffin IHC slide against the tissue IHC pattern and its controls. Record the ICC-IF observation in its own context; do not use intracellular ICC localization as the sole IHC scoring rule. |
Experiment evidence
IHC & IF images showing COL6A3 staining
A02844 · IHCImmunohistochemistry analysis of paraffin-embedded human breast carcinoma tissue, using Collagen VI alpha3 Antibody. The picture on the righ
A02844 · IFImmunofluorescence analysis of HeLa cells, using Collagen VI alpha3 Antibody. The picture on the right is blocked with the synthesized pepti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000163359-COL6A3/tissue/cerebral+cortex#imid_2804560
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000163359-COL6A3/tissue/colon#imid_2804470
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000163359-COL6A3/tissue/liver#imid_2804479
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000163359-COL6A3/tissue/kidney#imid_2804488
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA010007; green = COL6A3, blue = nucleus, red = microtubules.
HPA · ICC-IF ASC52teloHuman Protein Atlas immunofluorescence, ASC52telo, antibody HPA010007; green = COL6A3, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for COL6A3 IHC & IF
🧪Run ovary first and score ovarian stroma cells for staining (HPA: Medium in ovarian stroma cells). Use bronchus respiratory epithelial cells as the negative comparator (HPA: Not detected in respiratory epithelial cells); on the ovary slide, assess nonstromal cells as candidate internal negatives, while allowing for extracellular matrix staining around them (UniProt P12111: extracellular matrix localization).
Positive control tissue: Bone marrow (Hematopoietic cells, HPA Medium)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show COL6A3 in ASC52telo, BJ [Human fibroblast], U2OS, with annotated localisation: Endoplasmic reticulum (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and immunoglobulin class, or normal IgG from the same species for a polyclonal primary. A peptide-blocked section provides a biological specificity control (A02844 tissue-IHC caption: signal blocked with synthesized peptide); quench endogenous peroxidase for chromogenic detection and check ovarian tissue for background before scoring.
⚠️Feasibility: A target-specific fixation window and the effect of antigen retrieval are unreported in the supplied evidence; optimize retrieval empirically for paraffin sections. The selected A02844 tissue-IHC caption reports paraffin-embedded breast carcinoma but does not report its fixative (A02844 tissue-IHC caption). The evidence does not establish that frozen sections or IF are easier; score extracellular staining separately from cellular background (UniProt P12111: extracellular matrix localization).
HPA tissue IHC evidence for COL6A3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — Low consistency between antibody staining and RNA expression data. At least one protein variant secreted, tissue location of RNA and protein might differ and correlation is complex. Antibody staining in cells/structures not annotated, view images.). 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 | Medium | Protein (IHC) | HPA → |
| Ovary | Ovarian stroma cells | Medium | 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 COL6A3 IHC Tips
Troubleshoot COL6A3 staining in paraffin-section chromogenic IHC by assessing retrieval, tissue handling, extracellular localisation and compartment-specific scoring (UniProt P12111; HPA tissue IHC).
How should I optimize retrieval when COL6A3 staining is weak?
For paraffin-section IHC, start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval setting). Keep section thickness, cooling time and detection conditions constant while comparing retrieval runs, so a staining change can be attributed to retrieval (standard IHC practice). If staining remains weak, test a different retrieval condition on adjacent sections as a fallback, recording its buffer, pH, temperature and duration (standard IHC practice). Inspect extracellular matrix and tissue integrity together, because COL6A3 is secreted into the extracellular matrix and harsh retrieval can impair morphology (UniProt P12111 localisation; standard IHC practice).
Could fixation explain absent or uneven COL6A3 staining?
Target-specific fixation sensitivity is unknown from the supplied evidence; the selected A02844 image describes paraffin-embedded breast carcinoma but does not report its fixative (A02844 caption). Record fixative, fixation duration and processing history for each specimen before comparing staining intensity across sections (standard IHC practice). Process a small comparison set with matched section thickness, retrieval and detection conditions to determine whether local handling explains the difference (standard IHC practice). Check morphology and staining near section edges separately, since uneven processing can complicate interpretation of a secreted matrix protein (standard IHC practice; UniProt P12111 localisation).
Where should convincing COL6A3 staining appear in tissue?
Assess extracellular matrix staining first, because COL6A3 is a secreted extracellular matrix protein without a transmembrane segment (UniProt P12111 localisation and topology). Some cytoplasmic staining can be plausible: tissue IHC reports cytoplasmic expression in a few tissues with matrix positivity, while cell imaging places COL6A3 mainly in endoplasmic reticulum and additionally in vesicles (HPA tissue IHC; HPA subcellular). Compare stained structures with the corresponding morphology and document matrix and cellular signal separately rather than assigning both to one cell type (standard IHC practice). A diffuse nuclear-only pattern needs scrutiny against controls because it does not match the supplied localisation evidence (UniProt P12111 localisation; HPA subcellular).
How can isoforms or epitope accessibility change the IHC pattern?
COL6A3 has 5 annotated isoforms, so establish which sequence region the IHC-validated antibody recognizes before treating staining as total COL6A3 (UniProt P12111 isoforms; standard IHC practice). Its domains include repeated VWFA regions, collagen-like segments, a fibronectin type-III region and a BPTI/Kunitz region (UniProt P12111 domains). The record also lists glycosylation and modified residues, which makes epitope placement relevant when comparing processed tissue or antibody clones (UniProt P12111 glycosylation and modified residues; standard IHC practice). If the epitope is undisclosed, report the antibody and observed staining pattern without claiming isoform-specific detection or attributing weak signal to a particular modification (standard IHC practice).
How should I investigate COL6A3 by multiplex IF?
Use the separate IF/ICC workflow and pair COL6A3 with a marker for the expected cell type, while evaluating extracellular signal independently of cellular colocalisation (UniProt P12111 localisation; standard IF practice). Choose a fluorophore channel after inspecting unstained tissue autofluorescence, then include single-label controls to identify bleed-through in the multiplex image (standard IF practice). For an intracellular epitope, test gentle permeabilisation because cell imaging places COL6A3 in endoplasmic reticulum and vesicles (HPA subcellular; standard IF practice). For extracellular matrix detection, compare an otherwise matched condition without permeabilisation; COL6A3 lacks a transmembrane segment, so membrane-side assumptions require epitope information (UniProt P12111 topology and localisation; standard IF practice).
What should I check when matrix staining looks like background?
Include a no-primary control and inspect endogenous peroxidase activity before interpreting brown chromogen as COL6A3 signal (standard chromogenic IHC practice). Compare the stained section with its counterstained morphology: COL6A3 is extracellular, and tissue IHC reports matrix positivity alongside limited cytoplasmic expression (UniProt P12111 localisation; HPA tissue IHC). If broad staining obscures structures, adjust primary-antibody concentration or detection development using matched sections and keep retrieval fixed during that comparison (standard IHC practice). Record edge staining, folds, necrotic regions and nonspecific precipitate separately; those patterns can mimic a widespread matrix signal without identifying the antigen (standard IHC practice).
What is the most defensible way to quantify COL6A3 IHC? ⚠ ANSWER MARKED FOR VERIFICATION
Define compartments before scoring: measure extracellular positive area or optical density per mm² of evaluable matrix, and score cellular staining separately (UniProt P12111 localisation; standard image-analysis practice). An H-score or percentage of positive cells can summarize a defined cell population, but it does not capture extracellular deposition by itself (standard IHC scoring practice). Normalize matrix measurements to the annotated viable stromal or other relevant tissue area, and apply one threshold and imaging setup across comparison groups (standard image-analysis practice). Report section exclusions, compartment definitions and antibody identity, since secreted protein may be seen away from cells that made it (HPA tissue IHC reliability description; standard IHC practice).
How do I distinguish genuine COL6A3 signal from artefact?
Treat organised extracellular matrix staining as biologically plausible, while checking any cellular staining against morphology and the reported endoplasmic reticulum or vesicle localisation (UniProt P12111 localisation; HPA subcellular). HPA reports medium staining in ovarian stroma cells and bone-marrow hematopoietic cells, but warns that secreted protein can complicate correspondence between staining and RNA location (HPA tissue IHC). Reassess nuclear-only staining, section-edge gradients, necrotic debris and signal reproduced in a no-primary control before calling a region positive (UniProt P12111 localisation; standard IHC practice). Check endogenous peroxidase and chromogen deposits when apparent positive cells lack coherent anatomy, and report uncertain regions separately from interpretable staining (standard chromogenic IHC practice).
Boster reagents
Best COL6A3 / Collagen alpha-3(VI) chain IHC Antibodies
A02844 has IHC data from paraffin-embedded human breast carcinoma tissue and IF data from HeLa cells (catalog: image captions); listed reactivity is human and mouse (catalog: reactivity).
Real IHC data
Anti-Collagen VI alpha3 COL6A3 Antibody
Cat # A02844
A02844 was shown in IHC on paraffin-embedded human breast carcinoma tissue, with peptide-blocked comparison (catalog: IHC image caption). The same SKU was shown in IF on HeLa cells, with peptide-blocked comparison (catalog: IF image caption).
Which to pick: Choose A02844 for tissue IHC on paraffin sections, the preparation shown in its IHC image; the fixative is unreported (catalog: IHC image caption). Choose it for IF/ICC because both applications are listed and its IF image shows HeLa cells (catalog: applications and IF image caption). For human or mouse samples, it is the listed rabbit polyclonal option, although the supplied images show human samples only (catalog: host, polyclonal designation, reactivity and image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P12111 (CO6A3_HUMAN, Collagen alpha-3(VI) chain).
- Human Protein Atlas. COL6A3 tissue IHC expression (reliability: Supported).
- Human Protein Atlas. COL6A3 subcellular location (ICC-IF): Mainly localized to the endoplasmic reticulum. In addition localized to vesicles..
- Human Protein Atlas. COL6A3 antibody validation summary (2 antibodies).
- Tumor-specific expression and alternative splicing of the COL6A3 gene in pancreatic cancer. Surgery 2011 — PMC3163121.
- Prognostic value of COL6A3 in pancreatic adenocarcinoma. Annals of hepato-biliary-pancreatic surgery 2020 — PMC7061042.
- Integrated Ultrasound Localization Microscopy and Multi-Omics for Mechanism-Driven Stratification of Azoospermia. American journal of men's health 2026 — PMC13424786.
- Stroma derived COL6A3 is a potential prognosis marker of colorectal carcinoma revealed by quantitative proteomics. Oncotarget 2015 — PMC4745773.
- PubMed PMID:1689238 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15815621 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for COL6A3 (Collagen alpha-3(VI) chain). Expected localisation Extracellular matrix; some 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: A02844 at 1:100 - 1:300 (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 breast carcinoma tissue; fixative not specified (datasheet A02844); 2. Fixation: Image fixative and duration unreported (datasheet A02844); 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-COL6A3, 1:100 - 1:300 (datasheet A02844); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep formalin fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Secreted variants can uncouple tissue RNA and protein staining (HPA tissue IHC); Isoform / epitope: Five isoforms; mature chain starts at residue 26 (UniProt); Regulation: Tissue-enhanced RNA in smooth muscle (HPA tissue RNA). Evidence: UniProt P12111, HPA tissue IHC · Supported, HPA ICC-IF · Mainly localized to the endoplasmic reticulum. In addition localized…, Datasheet A02844, PMC3163121, PMC7061042. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/COL6A3/immunohistochemistry-ihc-guide.