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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). Ovarian stroma and bone marrow hematopoietic cells show medium staining (HPA tissue IHC); titrate the catalog antibody across 1:100–1:300 (datasheet).
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 with some cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining in a few tissues; ECM positivity (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 fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Secreted protein may stain away from its RNA source (HPA tissue IHC) | |
| Regulation | Tissue-enhanced RNA in smooth muscle (HPA tissue RNA) | |
| Isoform / epitope | 5 isoforms; check antibody epitope coverage (UniProt) |
Section 1
Recommended COL6A3 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol with published COL6A3 protocols for pancreatic tissue, mouse azoospermia models, and colorectal tissue (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); use each paper’s stated retrieval when reproducing its assay.
Section 2
What Is the Expected COL6A3 Staining Pattern?
COL6A3 is secreted into the extracellular matrix and has no transmembrane segment (UniProt P12111 topology). In paraffin-section IHC, expect matrix positivity with some cytoplasmic staining; HPA reports medium staining in ovarian stroma cells and bone-marrow hematopoietic cells (HPA tissue IHC). HPA rates the tissue pattern Supported but notes low consistency with RNA expression, partly because secreted protein can appear away from its producing cells (HPA tissue IHC).
| Matrix-associated chromogenic signal, with cytoplasmic staining in some cells. | This fits the reported extracellular-matrix positivity and limited cytoplasmic expression (HPA tissue IHC), and the secreted, extracellular location of COL6A3 (UniProt P12111). Judge signal against the tissue structures on the same section; secretion means the stained location need not identify the cell that made the protein (HPA tissue IHC). |
| Medium signal in ovarian stroma cells or bone-marrow hematopoietic cells. | These are the supplied positive IHC examples (HPA tissue IHC: Medium). Use the annotated cell populations when assessing a positive control; a tissue name alone does not imply that every cell should stain (HPA tissue IHC). |
| Predominantly nuclear signal, or a crisp cell-surface outline without matrix or plausible cytoplasmic signal. | That pattern conflicts with the reported tissue profile and extracellular location (HPA tissue IHC; UniProt P12111 topology). Review morphology and the negative-control section before assigning it to COL6A3; nonspecific staining or detection artefact is a possible explanation (general IHC practice). |
| Strong staining confined to a cell type reported as not detected, such as adipocytes in adipose tissue. | This differs from that cell-type annotation (HPA tissue IHC: Adipose tissue adipocytes, Not detected). Check for cross-reactivity or endogenous detection activity with appropriate controls (general IHC practice). Do not treat the annotation as proof that all structures in that tissue lack COL6A3 (HPA tissue IHC). |
| Weak haze across the section, or no signal in annotated positive cells. | Uniform haze is hard to assign to the reported matrix and cellular pattern (HPA tissue IHC). No signal in ovarian stroma or bone-marrow hematopoietic cells departs from the supplied medium-staining examples (HPA tissue IHC); check the IHC run and control tissue before calling the sample negative (general IHC practice). |
💡Expected COL6A3 appearanceCall a positive result when matrix-associated signal and plausible cytoplasmic staining accompany medium staining in annotated ovarian stroma or bone-marrow hematopoietic cells; isolated nuclear staining or uniform haze is suspect (HPA tissue IHC; UniProt P12111 topology; general IHC practice).
| Secretion and tissue assignment | COL6A3 is extracellular after secretion (UniProt P12111). HPA warns that RNA and protein locations can differ and reports low consistency between staining and RNA expression (HPA tissue IHC). Interpret deposits in their tissue context rather than using RNA abundance to predict a particular cell's IHC intensity. |
| Cell-type controls | HPA records medium staining in ovarian stroma cells and bone-marrow hematopoietic cells, but no detected staining in adipocytes from adipose tissue (HPA tissue IHC). These annotations support cell-type comparisons; they do not establish a uniformly positive or negative whole section. |
| Antibody validation | The supplied HPA IHC antibody, HPA010080, is Supported; HPA010007 has ICC approval but no supplied IHC status (HPA antibodies). Treat those application labels separately. The tissue profile itself is Supported despite HPA's stated RNA–protein consistency caveat (HPA tissue IHC). |
| Processing, variants, and epitope | The sequence has a signal peptide at residues 1–25, a chain at 26–3177, and five listed isoforms (UniProt P12111). The supplied evidence gives no antibody epitope or isoform coverage; do not infer which forms a tissue stain detects or predict a retrieval response from sequence features. |
| Antigen retrieval | Retrieval is a general paraffin-section IHC workflow variable (general IHC practice). Neither supplied source establishes COL6A3-specific fixation sensitivity or a preferred retrieval condition. Use the selected antibody's validated IHC procedure and judge any adjustment against a positive control (general IHC practice). |
| IF/ICC Q&A: should intracellular fluorescence match tissue IHC? | HPA ICC-IF places COL6A3 mainly in the endoplasmic reticulum, with vesicles also observed (HPA subcellular ICC-IF). Tissue IHC additionally shows matrix positivity (HPA tissue IHC). These application-specific observations can coexist with secretion (UniProt P12111); this IHC guide does not prescribe an IF/ICC protocol. |
| Situation | Likely cause | Next action |
|---|---|---|
| No chromogenic signal in the test section or annotated positive control. | The staining run may have failed; ovarian stroma and bone-marrow hematopoietic cells are medium-staining reference populations (HPA tissue IHC). | Confirm that the annotated cells are present, then check antibody application, retrieval, detection reagents, and control processing under the validated IHC procedure (general IHC practice). Avoid calling the test section negative until its positive control works. |
| Only nuclei stain strongly. | An exclusively nuclear pattern conflicts with extracellular COL6A3 and the reported matrix-positive tissue profile (UniProt P12111 topology; HPA tissue IHC). | Compare the signal with section morphology and a reagent negative control; review blocking and detection background before scoring it as specific (general IHC practice). |
| The whole section has diffuse chromogenic haze. | Widespread background can obscure the reported matrix and limited cytoplasmic pattern (HPA tissue IHC); inadequate blocking or washing can contribute (general IHC practice). | Check the reagent negative control, blocking, washes, and detection exposure, then reassess whether staining follows identifiable structures (general IHC practice). |
| Adipocytes stain strongly in an adipose-tissue section. | HPA reports those cells as not detected (HPA tissue IHC: Adipose tissue adipocytes); cross-reactivity or endogenous detection activity is possible (general IHC practice). | Compare a reagent negative control and inspect whether the deposit follows adipocytes rather than adjacent matrix (general IHC practice; UniProt P12111 extracellular location). Report the affected cell type precisely. |
| A low-staining cell population is scored as a strong positive control. | Smooth muscle cells are listed Low, while ovarian stroma cells and bone-marrow hematopoietic cells are Medium in the supplied tissue annotations (HPA tissue IHC). | Use the annotated medium-staining populations to assess run performance when available; score the actual cells and matrix separately (HPA tissue IHC; general IHC practice). |
| ICC-IF shows intracellular signal while tissue IHC emphasizes matrix. | HPA places the ICC-IF signal mainly in endoplasmic reticulum and also vesicles, while its tissue IHC profile includes extracellular matrix (HPA subcellular ICC-IF; HPA tissue IHC). | Interpret each image within its application and controls; secretion makes intracellular and extracellular observations compatible (UniProt P12111; HPA tissue IHC). Use the separate IF/ICC guide for IF-specific decisions. |
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 assess staining in ovarian stroma cells (HPA: Medium in ovarian stroma cells). Use adipose tissue as the negative comparison, scoring adipocytes, which are reported as not detected (HPA: Not detected in adipocytes); on the ovarian slide, assess non-stromal cells as potential internal negatives against the stained stroma rather than assuming they are negative (HPA: Medium in ovarian stroma cells).
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 section and an isotype control matched to the primary antibody’s host species, immunoglobulin class, and concentration (standard IHC practice). Add peptide blocking as a specificity control (A02844 caption: staining was blocked with synthesized peptide), and block endogenous peroxidase before chromogenic detection of ovarian sections (standard IHC practice).
⚠️Feasibility: The A02844 paraffin-section caption does not report a fixative, and the supplied evidence reports no target-specific fixation window or fixation effect (A02844 caption). Antigen-retrieval dependence is unreported; compare retrieval conditions on paraffin sections, while the supplied evidence does not establish whether frozen sections or IF/ICC are easier (A02844 caption; HPA subcellular: ICC-IF images). In ovary, score stromal extracellular staining separately from staining within cells so matrix next to a cell is not assigned to that cell (HPA: Medium in ovarian stroma cells; UniProt P12111: extracellular matrix).
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
Troubleshooting COL6A3 chromogenic IHC requires attention to extracellular matrix staining, intracellular synthesis, and the limits of the available antibody evidence.
How should I adjust retrieval when COL6A3 staining is weak or uneven?
For paraffin-section COL6A3 IHC, start with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval setting). Keep section thickness, heating, and cooling consistent across cases before attributing uneven staining to antigen abundance (standard IHC practice). If staining remains weak, test a modest increase in retrieval time on adjacent sections while watching for tissue detachment and loss of matrix detail (standard IHC practice). Compare changes in extracellular matrix staining as well as cellular staining, because COL6A3 is secreted into the matrix (UniProt P12111 localisation). A peptide-blocked image exists for catalog antibody A02844, but its caption does not establish a retrieval condition (A02844 tissue-IHC caption).
Could fixation explain variable COL6A3 staining between paraffin blocks?
Target-specific fixation sensitivity for COL6A3 is unknown from the supplied evidence; the catalog image identifies paraffin-embedded breast carcinoma but does not state its fixative (A02844 tissue-IHC caption). Record each block’s fixative and fixation duration when available, then compare sections processed with the same retrieval and detection conditions (standard IHC practice). Excessive or inconsistent fixation can alter antigen access in IHC generally, so use matched processing and an appropriate control section to investigate variability (standard IHC practice). Do not assign a COL6A3-specific fixation effect based on extracellular localisation, modifications, or HPA staining patterns (UniProt P12111 localisation and modifications; HPA tissue IHC).
Should COL6A3 appear in cells, extracellular matrix, or both?
Prioritise extracellular matrix staining when assessing COL6A3 in chromogenic sections, because the mature protein is secreted and has no transmembrane segment (UniProt P12111 localisation and topology). Intracellular signal can also be plausible during synthesis: HPA reports mainly endoplasmic reticulum localisation, with additional vesicular localisation in its subcellular assessment (HPA subcellular). HPA tissue IHC describes cytoplasmic expression in a few tissues alongside extracellular matrix positivity, so record the compartments separately (HPA tissue IHC). A membrane-only outline without accompanying plausible matrix or intracellular distribution deserves scrutiny for nonspecific staining, especially if it also appears in the negative control (UniProt P12111 topology; standard IHC practice).
Can an absent stain reflect isoform selection or an inaccessible epitope?
COL6A3 has 5 listed isoforms, but the supplied antibody caption does not identify an epitope or establish which isoforms catalog antibody A02844 detects (UniProt P12111 isoforms; A02844 tissue-IHC caption). Its sequence includes von Willebrand factor A, collagen-like, fibronectin type-III, and Kunitz domains, so antibody recognition cannot be inferred from the gene name alone (UniProt P12111 domains). Glycosylation and other modifications are annotated, but their effects on this antibody’s IHC staining are untested here (UniProt P12111 modifications; A02844 tissue-IHC caption). If results conflict with another assay, obtain the antibody’s immunogen location and isoform coverage before interpreting a negative section as absence of COL6A3 (standard IHC practice).
How can IF help resolve ambiguous COL6A3 IHC localisation?
Use IF as a separate localisation check and multiplex COL6A3 with a validated marker for the cell population under study, while examining extracellular signal independently of cell boundaries (UniProt P12111 localisation; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, and reserve a spectrally distinct channel for the weaker signal (standard IF practice). COL6A3 has no transmembrane segment; extracellular matrix epitopes may be accessible without permeabilisation, whereas intracellular endoplasmic reticulum or vesicle signal requires an appropriately permeabilised preparation (UniProt P12111 topology; HPA subcellular; standard IF practice). Include single-stain and secondary-only controls to separate channel bleed-through or background from a convincing COL6A3 pattern (standard IF practice).
What controls help distinguish COL6A3 staining from chromogenic background?
Inspect a no-primary control for secondary-reagent or detection background, and use an appropriate peroxidase block before chromogenic development (standard IHC practice). Compare suspected staining with the peptide-blocked image for catalog antibody A02844, recognising that its caption documents a comparison in paraffin-embedded breast carcinoma without reporting the fixative (A02844 tissue-IHC caption). Diffuse colour over damaged edges or necrotic regions should be assessed against intact tissue and the no-primary control before it is scored (standard IHC practice). Because genuine COL6A3 can occupy extracellular matrix, avoid dismissing all acellular staining as background; judge its distribution and control behaviour together (UniProt P12111 localisation; standard IHC practice).
How should I quantify matrix-rich COL6A3 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the tissue region and exclude folds, detached areas, and necrosis before measuring COL6A3 chromogenic signal (standard IHC practice). For extracellular staining, measure positive matrix area or stain density per mm² of evaluable tissue, and report the threshold and background subtraction method (UniProt P12111 localisation; standard image-analysis practice). If cellular staining is the question, score the percentage of positive cells or an H-score separately from matrix signal, using a prespecified cell compartment (HPA tissue IHC; standard IHC practice). Normalise each result to comparable evaluable area or counted cells, and keep acquisition and analysis settings fixed across groups (standard IHC practice).
When is a COL6A3-positive IHC result biologically convincing?
A convincing result has reproducible extracellular matrix staining, with any intracellular component assessed separately, consistent with COL6A3 secretion and HPA observations (UniProt P12111 localisation; HPA tissue IHC). HPA reports medium staining in ovarian stroma cells and bone marrow hematopoietic cells, while its tissue reliability is Supported with a warning that RNA and secreted protein locations may differ (HPA tissue IHC). Treat isolated nuclear or membrane-only colour, edge staining, and necrotic deposits cautiously, then check the no-primary control and peroxidase-block performance (UniProt P12111 topology; standard IHC practice). Peptide blocking supports antibody-dependent staining in the catalog image but cannot, by itself, establish the identity of every positive structure in a new specimen (A02844 tissue-IHC caption; standard IHC practice).
Boster reagents
Best COL6A3 / Collagen alpha-3(VI) chain IHC Antibodies
A02844 has IHC data from human breast carcinoma paraffin sections and IF data from HeLa cells (catalog image captions); its listed reactivity is human and mouse (catalog).
Real IHC data
Anti-Collagen VI alpha3 COL6A3 Antibody
Cat # A02844
A02844 is listed for IHC, IF, ICC and ELISA in human and mouse (catalog applications and reactivity). Its IHC image shows human breast carcinoma paraffin sections, and its IF image shows HeLa cells; both captions describe peptide-blocked comparison images (catalog image captions).
Which to pick: Choose A02844 for paraffin-section tissue IHC because its own IHC image shows human breast carcinoma paraffin sections; the fixative is unreported (catalog IHC image caption). Choose A02844 for IF/ICC because both applications are listed and its own IF image shows HeLa cells (catalog applications and IF image caption). For human or mouse samples, A02844 is the listed rabbit polyclonal option (catalog host, clonality and reactivity); the supplied images document human samples only (catalog 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 with some cytoplasmic 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 fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secreted protein may stain away from its RNA source (HPA tissue IHC); Isoform / epitope: 5 isoforms; check antibody epitope coverage (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.