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LAMA4 / Laminin subunit alpha-4 · IHC design guide
Design Immunohistochemistry for LAMA4
Plan chromogenic IHC for LAMA4 using its basement membrane and vascular cell staining profile (HPA tissue IHC). This guide covers fixation, controls and interpretation for a secreted extracellular matrix protein (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
LAMA4 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 | Basement membranes; endothelial and smooth muscle cells (HPA tissue IHC) | |
| Staining pattern | Basement membrane staining with endothelial and smooth muscle staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Smooth muscle+1 more · see all | |
| Negative control | Adrenal gland+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 protein may stain away from RNA-positive cells (HPA tissue IHC) | |
| Regulation | Expression varies between adult and fetal tissues (UniProt) | |
| Isoform / epitope | Three isoforms; signal peptide 1–24 is removed, so map the epitope (UniProt) |
Section 1
Recommended LAMA4 IHC & IF Protocols
The catalog antibody’s IHC-P protocol is paired with published chromogenic IHC methods for canine hemangiosarcoma and human tissue arrays (PMC7310061; PMC4875769).
| Sample | Paraffin-embedded human colorectal carcinoma tissue; fixative not specified (datasheet A04999-1) |
| Fixation | Image fixative and duration unreported (datasheet A04999-1); 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-LAMA4, 1:50-1:200 (datasheet A04999-1) |
| 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 | LAMA4-positive staining in smooth muscle cells of smooth muscle (HPA tissue IHC: High). HPA tissue profile: Distinct expression in endothelial cells, smooth muscle cells, basement membranes and decidual cells. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page antigen retrieval); the canine study used 40 min in citrate (PMC7310061: Methods).
Section 2
What Is the Expected LAMA4 Staining Pattern?
LAMA4 is a secreted basement membrane and extracellular matrix protein with no transmembrane segment (UniProt Q16363 topology). In paraffin section IHC, expect staining along basement membranes and in regions associated with endothelial and smooth muscle cells; decidual cells may also stain (HPA tissue IHC). HPA rates tissue IHC reliability as Enhanced while noting medium consistency with RNA data, partly because a secreted protein can appear away from its site of production (HPA tissue IHC).
| Basement membrane outlines stain near vessels or smooth muscle, with little nuclear signal. | This fits LAMA4's extracellular basement membrane location (UniProt Q16363 subcellular location) and HPA's distinct staining in basement membranes, endothelial cells and smooth muscle cells (HPA tissue IHC). Judge the pattern by its location as well as its intensity. |
| Smooth muscle cells stain strongly; decidual cells show a weaker positive pattern. | These are useful reference patterns: HPA reports High staining in smooth muscle cells and Medium staining in placental decidual cells (HPA tissue IHC). A weaker decidual result can still be consistent with the reference; assess it alongside the expected basement membrane distribution. |
| Signal is predominantly nuclear or confined to an unexpected compartment. | A predominantly nuclear IHC pattern conflicts with the secreted extracellular location and lack of a transmembrane segment (UniProt Q16363 topology). Treat it as suspect and review controls and detection background (general IHC practice). HPA reports plasma membrane and cytosol in ICC-IF, which is a separate application (HPA subcellular). |
| Strong staining appears in an unexpected cell population, such as adrenal glandular cells. | HPA reports adrenal glandular cells as Not detected (HPA tissue IHC). Check whether the signal follows a basement membrane rather than the cells themselves (UniProt Q16363 subcellular location). If it is truly cellular, assess antibody cross-reactivity and endogenous detection activity with appropriate controls (general IHC practice). |
| Diffuse haze obscures tissue boundaries, or a reference positive section has no specific signal. | Haze prevents a reliable location call; absent staining in smooth muscle cells also conflicts with an HPA High reference (HPA tissue IHC). Compare positive and negative controls, then inspect background, primary antibody dilution, retrieval and detection performance (general IHC practice). |
💡Expected LAMA4 appearanceCall an IHC result positive when basement membranes and endothelial or smooth muscle regions show a defined pattern, with High staining possible in smooth muscle cells and Medium staining in decidual cells (HPA tissue IHC); diffuse haze or predominantly nuclear signal does not match LAMA4's extracellular location (UniProt Q16363 subcellular location).
| Extracellular location | LAMA4 is secreted and is a major basement membrane component, without a transmembrane segment (UniProt Q16363 subcellular location and topology). Interpret staining in relation to tissue boundaries; an outline around cells need not mean that the outlined cells produced the protein (HPA tissue IHC reliability note). |
| Tissue reference and reliability | HPA reports High staining in smooth muscle cells, Medium staining in placental decidual cells and Not detected in several specified cell populations (HPA tissue IHC). Its Enhanced reliability carries a medium RNA–staining consistency caveat because secreted protein and RNA locations can differ (HPA tissue IHC). |
| Antibody validation | HPA lists HPA015693 and CAB078156 as IHC Enhanced (HPA antibodies). This supports comparison against HPA's tissue pattern, but it does not establish the performance of a different catalog antibody, a particular retrieval setting or fixation sensitivity (HPA antibodies; general IHC practice). |
| Processing and variants | UniProt lists a signal peptide at residues 1–24, a mature chain at 25–1823 and three isoforms (UniProt Q16363 processing and isoforms). If the catalog antibody's epitope is known, check its position against these annotations; epitope coverage is unknown from the supplied record. |
| IF/ICC question: Should its cell signal define the IHC result? | No. HPA reports approved plasma membrane and cytosol locations in ICC-IF images from A-431, U-251MG and U2OS (HPA subcellular). For paraffin section IHC, use the basement membrane and cell distribution reported in HPA tissue IHC; the applications need separate interpretation. |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in a smooth muscle reference section. | HPA reports High smooth muscle cell staining, so a blank result needs a technical check before interpretation (HPA tissue IHC). | Verify the positive control, primary antibody use, retrieval and chromogenic detection steps; adjust one general IHC variable at a time (general IHC practice). No LAMA4-specific retrieval condition is supplied. |
| Placental decidual staining is faint. | HPA reports Medium staining in decidual cells, below its High rating for smooth muscle cells (HPA tissue IHC). | Compare with a smooth muscle positive reference and check whether faint signal has the expected location before changing conditions (HPA tissue IHC; general IHC practice). |
| Whole-section brown haze hides basement membrane outlines. | Non-specific background or endogenous detection activity can obscure a localized chromogenic signal (general IHC practice). | Inspect a no-primary control, blocking and detection steps, then optimize primary antibody dilution against a positive reference (general IHC practice). |
| Strong glandular cell signal appears in adrenal gland or appendix. | HPA reports those glandular cell populations as Not detected; cross-reactivity or endogenous detection activity is possible (HPA tissue IHC; general IHC practice). | Check a no-primary control and the exact cellular location. Compare the pattern with an independently validated IHC antibody where available (general IHC practice; HPA antibodies). |
| Most of the apparent signal is nuclear. | That compartment conflicts with UniProt's extracellular basement membrane annotation (UniProt Q16363 subcellular location). | Check the no-primary control, counterstain and chromogenic detection background before assigning LAMA4 positivity (general IHC practice). |
| IHC and ICC-IF appear to show different locations. | HPA reports basement membrane and tissue-associated IHC staining, but approved plasma membrane and cytosol locations in ICC-IF (HPA tissue IHC; HPA subcellular). | Interpret each result within its application and controls. Do not use the ICC-IF location alone to score paraffin section IHC (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing LAMA4 staining
A04999-1 · IHCImmunohistochemistry (IHC) analyzes of Laminin α-4 (A511) pAb in paraffin-embedded human colorectal carcinoma tissue at 1:50.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000112769-LAMA4/tissue/cerebral+cortex#imid_20124405
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000112769-LAMA4/tissue/colon#imid_20124323
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000112769-LAMA4/tissue/liver#imid_20124340
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000112769-LAMA4/tissue/kidney#imid_20124333
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA015693; green = LAMA4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA015693; green = LAMA4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for LAMA4 IHC & IF
🧪Run smooth muscle first and assess staining associated with smooth muscle cells (HPA: High in smooth muscle cells). Use adrenal gland glandular cells as a negative comparator (HPA: Not detected in adrenal gland glandular cells); on the positive slide, treat neighboring cells without specific signal as internal background comparators, while accounting for extracellular basement-membrane staining around cells (UniProt Q16363 subcellular).
Positive control tissue: Smooth muscle (Smooth muscle cells, HPA High)
Negative control tissue: Adrenal gland (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show LAMA4 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and a control immunoglobulin matched to the primary antibody’s host species, isotype or clonality, and concentration; use LAMA4 knockout material or validated immunogen-peptide competition as a biological specificity control (standard IHC practice). For chromogenic detection, check endogenous peroxidase and biotin background on the positive tissue (standard IHC practice).
⚠️Feasibility: The supplied target and application evidence reports no LAMA4-specific fixation window, fixation effect, or antigen-retrieval dependency; optimize retrieval empirically (supplied evidence; standard IHC practice). The selected A04999-1 paraffin-section caption reports a 1:50 dilution but leaves the fixative unreported (selected tissue-IHC caption). The evidence does not establish that frozen sections or IF are easier; interpret signal by its extracellular matrix and basement-membrane distribution, which can border cells (UniProt Q16363 subcellular).
HPA tissue IHC evidence for LAMA4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium 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 |
|---|---|---|---|---|
| Smooth muscle | Smooth muscle cells | High | Protein (IHC) | HPA → |
| Placenta | Decidual cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic 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 LAMA4 IHC Tips
Troubleshoot LAMA4 chromogenic IHC by assessing basement membrane staining alongside cell type, tissue architecture, and controls (UniProt Q16363 localisation; HPA tissue IHC).
How should I optimize antigen retrieval when LAMA4 staining is weak?
Start with citrate buffer at pH 6.0, heated to 95–98 °C for 20 minutes, for paraffin section chromogenic IHC (page retrieval rule). Compare serial sections using the same antibody dilution, detection chemistry, and exposure to the chromogen so retrieval is the main variable (standard IHC practice). If staining remains weak, test a different retrieval buffer or heating duration on adjacent sections while checking for tissue damage (standard IHC practice). Assess basement membranes and smooth muscle structures as reference locations, rather than judging retrieval solely by diffuse cellular color (UniProt Q16363 localisation; HPA tissue IHC).
Can fixation explain weak or uneven LAMA4 staining?
Target specific fixation sensitivity is unknown because the supplied tissue IHC caption identifies paraffin embedded material but does not report a fixative (caption A04999-1). Record the fixative, fixation duration, section thickness, and processing history for each specimen before comparing staining intensity (standard IHC practice). On serial sections, hold retrieval at citrate pH 6.0 and 95–98 °C for 20 minutes while comparing specimens with documented processing (page retrieval rule; standard IHC practice). Check morphology and staining at tissue edges as well as central regions; uneven processing can complicate interpretation without establishing a LAMA4 specific fixation effect (standard IHC practice).
Where should convincing LAMA4 chromogenic staining appear?
Prioritize staining along basement membranes and other extracellular matrix structures because LAMA4 is secreted and is a major basement membrane component (UniProt Q16363 localisation). Endothelial cells and smooth muscle cells are useful anatomical reference points, while decidual cells are another reported tissue context (HPA tissue IHC). Do not require a crisp intracellular pattern to call a section positive: the protein lacks a transmembrane segment, and secreted protein can lie apart from its producing cell (UniProt Q16363 topology; HPA tissue IHC reliability description). Compare staining with a matched negative control and the section’s architecture before assigning signal to individual cells (standard IHC practice).
Could isoforms or epitope accessibility change the LAMA4 IHC pattern?
LAMA4 has 3 listed isoforms, but the supplied antibody evidence does not identify which isoforms its epitope detects (UniProt Q16363 isoforms; caption A04999-1). Map any disclosed immunogen to the 25–1823 mature chain before interpreting absent staining as absent total LAMA4 (UniProt Q16363 processing; standard IHC practice). The protein has 20 listed glycosylation sites and multiple laminin EGF like and G like domains, which makes epitope location relevant to antibody comparison without proving a staining effect (UniProt Q16363 glycosylation and domains). Compare antibodies on serial sections only after documenting their epitopes, retrieval conditions, and detection systems (standard IHC practice).
How can IF help resolve ambiguous LAMA4 tissue staining?
For a separate IF experiment, pair LAMA4 with an endothelial marker such as CD31 or a smooth muscle marker such as α smooth muscle actin, guided by the reported tissue pattern (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores and consider a far red LAMA4 channel when tissue autofluorescence obscures shorter wavelengths (standard IF practice). For an extracellular epitope, begin without detergent permeabilisation; assess gentle permeabilisation only if the antibody’s documented epitope requires intracellular access (UniProt Q16363 topology and localisation; standard IF practice). Include single stain and no primary controls, and do not carry the paraffin IHC caption’s 1:50 dilution into IF without validation (caption A04999-1; standard IF practice).
What controls help distinguish LAMA4 signal from chromogenic background?
Run a no primary control through the same peroxidase detection and DAB development steps to reveal secondary reagent or endogenous enzyme background (standard IHC practice). Include a peroxidase block and compare staining after identical DAB development times across sections (standard IHC practice). Examine pigment, folds, detached tissue, and section edges before attributing brown deposits to LAMA4 (standard IHC practice). Use basement membrane associated staining and the reported smooth muscle or endothelial distribution as anatomical checks, while recognizing that secreted protein need not match cellular RNA locations exactly (UniProt Q16363 localisation; HPA tissue IHC and reliability description).
How should LAMA4 staining be scored across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define compartments before scoring: measure basement membrane positive length or stained area per mm² of evaluable tissue, and score cellular staining separately if it is present (UniProt Q16363 localisation; standard IHC practice). Normalize the matrix measure to available vessel perimeter or basement membrane length where anatomy permits, rather than comparing raw brown area between differently structured sections (standard IHC practice). For a clearly defined cell population, report the percentage positive and intensity or an H score, with thresholds fixed before comparison (standard IHC practice). Exclude folds, necrosis, and section edges consistently, and record retrieval and DAB conditions for every batch (standard IHC practice).
When is a positive LAMA4 IHC result biologically credible?
A credible result follows basement membrane or extracellular structures and is coherent with endothelial or smooth muscle anatomy (UniProt Q16363 localisation; HPA tissue IHC). Treat isolated nuclear staining or uniform cytoplasmic staining across unrelated cells cautiously because the protein is secreted and has no transmembrane segment (UniProt Q16363 topology and localisation). Check whether strong color is confined to section edges, necrotic regions, or sites that remain colored in a no primary control (standard IHC practice). Interpret absent glandular staining in context: several glandular cell populations were reported as not detected, while HPA describes only medium consistency between staining and RNA data (HPA tissue IHC and reliability description).
Boster reagents
Best LAMA4 / Laminin subunit alpha-4 IHC Antibodies
A04999-1 has IHC data from a paraffin-embedded human colorectal carcinoma section (IHC image caption); IF is a listed application, with Human, Mouse and Rat reactivity (catalog applications and reactivity).
Real IHC data
Anti-Laminin Alpha-4 (A511) LAMA4 Antibody
Cat # A04999-1
A04999-1 was shown in IHC on paraffin-embedded human colorectal carcinoma tissue at 1:50 (IHC image caption). IF is listed without an IF figure, and Human, Mouse and Rat are listed as reactive species (catalog applications and reactivity; catalog IF images).
Which to pick: For paraffin-section IHC, choose A04999-1: its polyclonal antibody has an IHC image from paraffin-embedded human colorectal carcinoma tissue (IHC image caption). For IF/ICC, A04999-1 lists IF, but provides no IF figure or separate ICC validation (catalog applications and IF images). For cross-species work, A04999-1 lists Human, Mouse and Rat reactivity; the pictured IHC sample is human, and its fixative is unreported (catalog reactivity; IHC image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q16363 (LAMA4_HUMAN, Laminin subunit alpha-4).
- Human Protein Atlas. LAMA4 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. LAMA4 subcellular location (ICC-IF): Localized to the plasma membrane and cytosol..
- Human Protein Atlas. LAMA4 antibody validation summary (2 antibodies).
- Extracellular matrix-related genes-based prognostic signature for cervical cancer: association of LAMA4 expression with prognosis and response to immunotherapy. Frontiers in oncology 2025 — PMC12380540.
- Chromatin run-on sequencing analysis finds that ECM remodeling plays an important role in canine hemangiosarcoma pathogenesis. BMC veterinary research 2020 — PMC7310061.
- MCAM and LAMA4 Are Highly Enriched in Tumor Blood Vessels of Renal Cell Carcinoma and Predict Patient Outcome. Cancer research 2016 — PMC4875769.
- Effect of Laminin-A4 inhibition on cluster formation of human osteoarthritic chondrocytes. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 2016 — PMC5727909.
- PubMed PMID:7781776 — UniProt-cited evidence.
- PubMed PMID:8706685 — UniProt-cited evidence.
- PubMed PMID:14574404 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for LAMA4 (Laminin subunit alpha-4). Expected localisation Basement membranes; endothelial and smooth muscle cells (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A04999-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Smooth muscle (Smooth muscle cells); negative Adrenal gland. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colorectal carcinoma tissue; fixative not specified (datasheet A04999-1); 2. Fixation: Image fixative and duration unreported (datasheet A04999-1); 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-LAMA4, 1:50-1:200 (datasheet A04999-1); 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 protein may stain away from RNA-positive cells (HPA tissue IHC); Isoform / epitope: Three isoforms; signal peptide 1–24 is removed, so map the epitope (UniProt); Regulation: Expression varies between adult and fetal tissues (UniProt). Evidence: UniProt Q16363, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and cytosol., Datasheet A04999-1, PMC7310061, PMC4875769. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/LAMA4/immunohistochemistry-ihc-guide.