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INSL3 / Insulin-like 3 · IHC design guide
Design Immunohistochemistry for INSL3
Plan chromogenic IHC on paraffin sections using testis Leydig cells as the positive reference (HPA tissue IHC). Compare cytoplasmic staining with controls, accounting for INSL3 secretion and processing (HPA tissue IHC; UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
INSL3 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 | Leydig cell cytoplasm (HPA tissue IHC) | |
| Staining pattern | Selective cytoplasmic staining in Leydig cells (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Testis | |
| 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 | Secretion may separate protein staining from its cellular source (HPA tissue IHC; UniProt) | |
| Regulation | Prenatal and postnatal Leydig cell expression (UniProt) | |
| Isoform / epitope | 2 isoforms; signal peptide and propeptide processing make epitope choice relevant (UniProt) |
Section 1
Recommended INSL3 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet) is accompanied by four published INSL3 IHC methods (PMC8382946; PMC12420327; PMC4808909; PMC8072358).
| Sample | Tissue sections; selected-image fixative not specified (standard IHC workflow) |
| Fixation | Image fixative and duration unreported (datasheet A01712); 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-INSL3, 1:50 - 1:100 (datasheet A01712) |
| 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 | INSL3-positive staining in leydig cells of testis (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic expression in Leydig cells in testis. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 HIER at 95–98 °C for 20 min (page retrieval); the human gonadal protocol also uses citrate retrieval (PMC8382946).
Section 2
What Is the Expected INSL3 Staining Pattern?
INSL3 is a secreted protein without a transmembrane segment (UniProt P51460 topology). In paraffin-section IHC, expect selective cytoplasmic staining of testicular Leydig cells, with a High staining level in the HPA reference tissue (HPA: testis, Leydig cells, High). HPA rates this tissue pattern Enhanced because staining is consistent with RNA expression; it cautions that a secreted protein's RNA and protein locations may differ (HPA: tissue IHC).
| Selective cytoplasmic signal in cells between testicular tubules. | This fits the reported Leydig-cell pattern when cell identity is established from morphology and tissue context (HPA: selective cytoplasmic expression in Leydig cells). Compare the distribution and intensity with a known-positive testis section; HPA reports High staining in Leydig cells, but that rating does not set an intensity threshold for every specimen (HPA: testis, Leydig cells, High). |
| Predominantly nuclear or membrane-restricted signal in the presumed positive cells. | That compartment differs from HPA's observed cytoplasmic tissue pattern (HPA: tissue IHC). INSL3 is secreted and has no transmembrane segment, so a crisp membrane-only pattern needs independent verification before it is scored as target staining (UniProt P51460 topology). Check morphology, the no-primary control and detection background; an unexpected compartment alone does not identify the cause (general IHC practice). |
| Strong signal in cells outside the expected Leydig-cell population. | First confirm cell identity on the counterstained section: HPA describes the testis pattern as selective for Leydig cells (HPA: tissue IHC). Staining elsewhere could reflect nonspecific antibody binding or detection activity, but should not be assigned either cause from appearance alone (general IHC practice). Review a no-primary control and a second specificity control before extending the cell-type interpretation. |
| Diffuse colour across stroma, tubules and section edges. | A broad, poorly cell-resolved deposit does not resemble selective Leydig-cell cytoplasm (HPA: tissue IHC). In chromogenic IHC, incomplete blocking, inadequate washing or detection background can produce diffuse colour (general IHC practice). Judge whether the same distribution appears in a no-primary control; background that obscures cell boundaries prevents a reliable call even if some Leydig cells look darker. |
| No discernible signal in morphologically identifiable Leydig cells. | A negative testis slide is discordant with the HPA reference pattern of High Leydig-cell staining (HPA: testis, Leydig cells, High). Confirm the section contains identifiable Leydig cells, then assess staining controls and the validated antibody workflow (general IHC practice). A failed positive control makes the run inconclusive; absence of colour in one specimen does not establish absence of INSL3. |
💡Expected INSL3 appearanceCall a convincing positive when High, selective cytoplasmic colour marks identifiable testicular Leydig cells; widespread, nuclear or membrane-only colour needs verification (HPA: testis tissue IHC; UniProt P51460 topology).
| Secretion and location | UniProt classifies INSL3 as secreted, while HPA observes cytoplasmic staining in Leydig cells (UniProt P51460 subcellular location; HPA: tissue IHC). Interpret tissue colour as an observed distribution, not proof that all INSL3 stays inside those cells. HPA explicitly notes that RNA and protein locations may differ for a secreted protein (HPA: reliability description). |
| Protein processing | The precursor has a signal sequence and a propeptide, and the functional subunit is described as linked A and B chains (UniProt P51460 processing and subunit). The supplied record does not locate the IHC antibody's epitope. Its recognition of precursor versus processed protein therefore cannot be inferred from processing annotations; consult epitope documentation before explaining a discrepant result. |
| Antibody evidence | HPA lists antibody HPA028615 as IHC Enhanced, and its tissue profile describes selective Leydig-cell cytoplasm (HPA: antibodies; HPA: tissue IHC). That supports the reported IHC pattern, without establishing how another antibody behaves or validating a specific ICC-IF distribution. HPA provides no ICC-IF images or main subcellular location for this target (HPA: subcellular). |
| Choice of comparison tissue | HPA reports High staining in testicular Leydig cells and Not detected in specified populations including adrenal glandular cells and adipose-tissue adipocytes (HPA: tissue IHC). Use the named cell populations when comparing sections; these entries do not establish that every cell in those tissues is negative. UniProt also lists expression outside testis (UniProt P51460 tissue specificity). |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected Leydig-cell signal is absent. | The positive tissue may be missing identifiable Leydig cells, or the staining run may have failed; the slide alone cannot distinguish these possibilities (HPA: testis, Leydig cells, High; general IHC practice). | Locate Leydig cells on the counterstain and review a known-positive testis section run alongside the sample (HPA: testis tissue IHC). If the control also fails, review the validated antibody dilution, retrieval and detection steps as general IHC troubleshooting. |
| Colour appears throughout the section. | The distribution is broader than HPA's selective Leydig-cell pattern (HPA: tissue IHC). Nonspecific detection or inadequate blocking and washing are possible general IHC causes. | Compare a no-primary control, then inspect blocking, washing and chromogen development (general IHC practice). Score INSL3 only where cell-resolved staining can be distinguished from the background (HPA: selective Leydig-cell pattern). |
| Strong colour appears in an unexpected testicular cell population. | Cell misidentification, antibody cross-reactivity or endogenous detection activity are possibilities (general IHC practice); HPA reports selective Leydig-cell cytoplasm in testis (HPA: tissue IHC). | Recheck morphology and counterstain, inspect the no-primary control and seek independent antibody or orthogonal evidence before assigning that population as INSL3 positive (general IHC practice). |
| Signal is mainly nuclear or outlines cell membranes. | That appearance conflicts with the HPA cytoplasmic observation; membrane-only staining also lacks support from INSL3's no-transmembrane topology (HPA: tissue IHC; UniProt P51460 topology). | Check focus, section morphology and control staining, then verify the unexpected compartment independently (general IHC practice). Do not use compartment alone to diagnose a particular staining failure. |
| A comparison tissue shows unexpected positive cells. | HPA's Not detected calls apply to named cell populations, such as adrenal glandular cells and adipose-tissue adipocytes, rather than every cell in either tissue (HPA: tissue IHC). | Identify the stained cell type before comparing it with HPA, and examine a no-primary control if its staining is unexpected (general IHC practice). Interpret the result against the population-specific HPA entry. |
| IF/ICC Q: Does a fluorescent compartment pattern confirm INSL3? | HPA reports no ICC-IF images or main subcellular location, and lists no ICC status for HPA028615 (HPA: subcellular; HPA: antibodies). | A: No compartment-specific IF/ICC pattern is established by these payloads. Use an independently validated IF/ICC assay and its controls to interpret fluorescence; the HPA Leydig-cell observation is tissue IHC evidence (HPA: tissue IHC; general IF practice). |
Experiment evidence
IHC & IF images showing INSL3 staining
A01712 · IHCImmunohistochemistry analysis of paraffinembedded Human prostate using INSL3 pAb at dilution of 1:100 (40xlens).
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/cerebral+cortex#imid_7109920
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/colon#imid_7109838
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/liver#imid_7109839
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000248099-INSL3/tissue/kidney#imid_7109832
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for INSL3 IHC & IF
🧪Run testis first: Leydig cells should stain strongly (HPA: High in Leydig cells). Use adipose tissue as a negative tissue (HPA: Not detected in adipocytes); on the testis slide, neighboring non-Leydig cells should lack comparable staining.
Positive control tissue: Testis (Leydig cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for INSL3; derive a cell-line control from the positive tissue's cell type (Leydig cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary (secondary-only) control, an isotype control matched to the primary antibody’s host species and immunoglobulin class, and a biological negative such as INSL3-knockout material or a peptide block if available. For chromogenic testis IHC, block endogenous peroxidase and check for background staining; for IF, assess tissue autofluorescence.
⚠️Feasibility: A target-specific fixation window and antigen-retrieval dependency are unreported in the supplied evidence; the selected A01712 paraffin-section caption does not state a fixative (caption: fixative not stated). There is no supplied basis to judge frozen sections or IF easier, and HPA lists no ICC-IF images (HPA subcellular: no cell lines with ICC-IF images). Because INSL3 is secreted (UniProt P51460: Secreted), interpret diffuse staining cautiously and assess interstitial background against the controls.
HPA tissue IHC evidence for INSL3
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Secreted protein, tissue location of RNA and protein is expected to differ.). 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 |
|---|---|---|---|---|
| Testis | Leydig cells | High | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced INSL3 IHC Tips
Troubleshoot INSL3 chromogenic IHC in paraffin sections using the specified retrieval conditions and cell-specific controls; the IF entry addresses multiplex imaging.
Which retrieval conditions should I try first for weak INSL3 staining?
Start with citrate buffer at pH 6.0 for heat-induced retrieval at 95–98 °C for 20 min in paraffin-section IHC (page retrieval specification). Allow sections to cool in buffer, then compare staining with a concurrently processed testis control containing Leydig cells (standard IHC practice; HPA: high in testis Leydig cells). If staining remains weak, test a different retrieval buffer on adjacent sections as a fallback while holding antibody concentration and detection constant (standard IHC practice). Judge improvement by selective Leydig-cell cytoplasmic signal rather than stronger staining everywhere, because that is the documented tissue pattern (HPA: selective cytoplasmic expression in Leydig cells).
Could fixation explain weak or uneven INSL3 staining?
Target-specific sensitivity of INSL3 to fixation is unknown from the supplied evidence; the selected A01712 caption identifies paraffin-embedded human prostate but does not state its fixative (A01712 tissue-IHC caption). Record the actual fixative and fixation duration for each specimen, and compare sections processed under matched conditions before changing antibody concentration (standard IHC practice). Include testis with Leydig cells as a positive control and examine morphology for uneven processing or tissue loss (HPA: high in testis Leydig cells; standard IHC practice). If a new fixation condition weakens staining, verify the change experimentally; neither HPA staining nor INSL3 topology establishes a fixation mechanism (HPA: tissue IHC; UniProt P51460 topology).
Where should convincing INSL3 staining appear in tissue sections?
Expect selective cytoplasmic staining in testis Leydig cells when assessing the documented tissue pattern (HPA: selective cytoplasmic expression in Leydig cells). INSL3 is secreted and has no transmembrane segment, so diffuse extracellular material alone cannot identify the producing cell (UniProt P51460: subcellular location and topology). Compare cell identity and staining distribution against adjacent morphology, using a concurrently stained testis section as the positive reference (HPA: high in testis Leydig cells; standard IHC practice). The A01712 image caption documents staining of paraffin-embedded human prostate at 1:100, but it does not establish that prostate staining should follow the Leydig-cell pattern (A01712 tissue-IHC caption).
How could processing or isoforms affect antibody staining?
INSL3 has 2 annotated isoforms and a 131-aa precursor with a signal segment at residues 1–20 and a propeptide at 58–104 (UniProt P51460: isoforms and processing). Its mature hormone comprises A and B chains linked by 2 disulfide bonds, so precursor and processed forms may present different epitopes (UniProt P51460: subunit and processing). Check the catalog antibody’s stated immunogen or epitope before attributing a staining difference to isoform expression (standard IHC practice). Without an epitope map or isoform-specific validation in the supplied evidence, IHC staining cannot distinguish the two isoforms or prove recognition of the mature hormone (UniProt P51460: isoforms; supplied antibody evidence).
How should I adapt INSL3 detection for multiplex IF?
For multiplex IF, pair INSL3 with a validated Leydig-cell marker and assess whether signals occupy the same cells in testis (HPA: selective cytoplasmic expression in Leydig cells; standard IF practice). Select fluorophores after imaging unstained tissue for autofluorescence, and favor channels with clear separation from that background (standard IF practice). INSL3 is secreted without a transmembrane segment; if the antibody targets an intracellular precursor epitope, use a mild permeabilisation condition and compare it with an unpermeabilised section (UniProt P51460: secretion and topology; standard IF practice). The supplied A01712 evidence is paraffin tissue IHC, so establish IF specificity independently with secondary-only and expected-cell controls (A01712 tissue-IHC caption; standard IF practice).
How can I reduce diffuse or misleading brown signal?
First compare a no-primary control with the stained section to identify detection-system background, and inspect tissue edges separately (standard chromogenic IHC practice). For peroxidase-based detection, apply a peroxidase block and assess DAB development under matched conditions; these are general workflow steps, not INSL3-specific validation (standard chromogenic IHC practice). Titrate the primary antibody around the documented 1:100 A01712 prostate-caption dilution on adjacent sections, recording whether background falls while cell-associated signal remains (A01712 tissue-IHC caption; standard IHC practice). Favor selective cytoplasmic staining in Leydig cells over broad tissue-wide color, and use an expected-negative tissue region only when its cell identity is clear (HPA: Leydig-cell pattern; standard IHC practice).
What should I score when comparing INSL3 IHC across sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the analysis compartment before scoring: count morphologically identified Leydig cells in testis and record the percentage positive, or apply a prespecified H-score combining staining intensity and positive-cell fraction (HPA: Leydig-cell pattern; standard IHC practice). Normalize positive counts to total evaluable Leydig cells, or report positive-cell density per mm² of evaluable tissue when comparing areas (standard IHC practice). Keep retrieval, antibody dilution, DAB development, imaging, and threshold settings consistent across compared sections (standard IHC practice). Report excluded damaged or necrotic regions and include the same positive-control tissue across runs so changes in staining can be separated from processing variation (standard IHC practice).
How do I distinguish genuine INSL3 staining from artefact?
A credible testis result shows cell-associated cytoplasmic signal in morphologically identified Leydig cells, matching the documented selective pattern (HPA: selective cytoplasmic expression in Leydig cells). Treat staining confined to nuclei, tissue edges, necrotic areas, or unrelated cell populations as suspect until corroborated with controls and morphology (HPA: Leydig-cell pattern; standard IHC practice). Compare no-primary and peroxidase-block controls when brown deposits could reflect detection background or endogenous enzyme activity (standard chromogenic IHC practice). Because INSL3 is secreted, extracellular signal can differ from the location of expression; it should not by itself establish which cell produced the protein (UniProt P51460: secreted; HPA: reliability description).
Boster reagents
Best INSL3 / Insulin-like 3 IHC Antibodies
A01712 has real IHC data from a human prostate paraffin section (catalog image caption). IF/ICC is listed for human, mouse, and rat, with no IF image supplied (catalog applications, reactivity, image alts).
Real IHC data
Anti-Insulin-like 3 INSL3 Antibody
Cat # A01712
A01712 is listed for IHC-P and IF/ICC in human, mouse, and rat (catalog applications, reactivity). Its image shows IHC on a human prostate paraffin section at 1:100; no IF image is supplied (catalog image caption, IF image alts).
Which to pick: Choose A01712 for paraffin-section tissue IHC because its own image documents that preparation in human prostate; the fixative is unreported (catalog image caption). Choose A01712 for IF/ICC because those applications are listed, although no IF image is supplied (catalog applications, IF image alts). For mouse or rat samples, A01712 lists reactivity with both species, but its IHC image shows human tissue only (catalog reactivity, image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P51460 (INSL3_HUMAN, Insulin-like 3).
- Human Protein Atlas. INSL3 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. INSL3 subcellular location (ICC-IF): Secreted.
- Human Protein Atlas. INSL3 antibody validation summary (1 antibodies).
- Serum Concentrations and Gonadal Expression of INSL3 in Eighteen Males With 45,X/46,XY Mosaicism. Frontiers in endocrinology 2021 — PMC8382946.
- Insulin-like peptide 3 is not a biomarker for pancreatic ductal adenocarcinoma. Frontiers in endocrinology 2025 — PMC12420327.
- Effects of in Utero Exposure to Dicyclohexyl Phthalate on Rat Fetal Leydig Cells. International journal of environmental research and public health 2016 — PMC4808909.
- The Production of Testosterone and Gene Expression in Neonatal Testes of Rats Exposed to Diisoheptyl Phthalate During Pregnancy is Inhibited. Frontiers in pharmacology 2021 — PMC8072358.
- PubMed PMID:8034302 — UniProt-cited evidence.
- PubMed PMID:8020942 — UniProt-cited evidence.
- PubMed PMID:7852540 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for INSL3 (Insulin-like 3). Expected localisation Leydig cell cytoplasm (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A01712 at 1:50 - 1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Leydig cells); negative Adipose tissue. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Tissue sections; selected-image fixative not specified (standard IHC workflow); 2. Fixation: Image fixative and duration unreported (datasheet A01712); 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-INSL3, 1:50 - 1:100 (datasheet A01712); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Secretion may separate protein staining from its cellular source (HPA tissue IHC; UniProt); Isoform / epitope: 2 isoforms; signal peptide and propeptide processing make epitope choice relevant (UniProt); Regulation: Prenatal and postnatal Leydig cell expression (UniProt). Evidence: UniProt P51460, HPA tissue IHC · Enhanced, HPA ICC-IF · Secreted, Datasheet A01712, PMC8382946, PMC12420327. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/INSL3/immunohistochemistry-ihc-guide.