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CLN5 / Bis(monoacylglycero)phosphate synthase CLN5 · IHC design guide
Design Immunohistochemistry for CLN5
Plan CLN5 chromogenic IHC in paraffin sections using the cytoplasmic tissue pattern reported by HPA (HPA tissue IHC). Compare staining across consistently fixed samples, and interpret intensity with HPA’s low staining–RNA concordance in mind (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CLN5 Immunohistochemistry Experimental Design Guide
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
Must know before staining
| Expected localisation | Cytoplasmic in tissue (HPA tissue IHC); lysosomal membrane expected (UniProt) | |
| Staining pattern | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Caudate+4 more · see all | |
| Negative control | Lymph node+3 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04893-2) | |
| Caveat | Low staining–RNA concordance complicates interpretation (HPA tissue IHC) | |
| Regulation | No specific staining regulator reported (UniProt) | |
| Isoform / epitope | No isoforms; cytoplasmic N-terminus absent from 44–358 form (UniProt) |
Section 1
Recommended CLN5 IHC & IF Protocols
The catalog antibody provides the paraffin-section protocol (datasheet). A published sheep-brain CLN5 IHC method provides additional chromogenic staining conditions (PMC10628725).
| Sample | Paraffin-embedded human appendix tissue; fixative not specified (datasheet A04893-2) |
| Fixation | Image fixative and duration unreported (datasheet A04893-2); 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-CLN5, 1:50-1:200 (datasheet A04893-2) |
| 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 | CLN5-positive staining in glial cells of caudate (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
💡Decision noteFor paraffin sections, start with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule). The sheep method does not specify retrieval (PMC10628725).
Section 2
What Is the Expected CLN5 Staining Pattern?
CLN5 is a lysosomal protein associated with membranes; residues 24–40 form a transmembrane segment, with residues 41–358 facing the lumen (UniProt O75503 topology). In paraffin sections, expect cytoplasmic staining, including strong signal in lung macrophages, cerebral cortex glial cells, and duodenal glandular cells (HPA: tissue IHC). HPA rates its IHC staining Approved but reports low consistency with RNA expression (HPA: reliability).
| Granular cytoplasmic staining in lung macrophages, with stronger signal than nearby unstained cells (HPA: High in lung macrophages). | This fits the reported cell pattern and is compatible with lysosomal localisation (HPA: tissue IHC; UniProt O75503 subcellular location). Granules alone do not prove lysosomal identity; compare cell morphology and controls (general IHC practice). |
| Predominantly nuclear staining, especially when cytoplasmic signal is absent (HPA: cytoplasmic expression in most tissues). | A nuclear-only result conflicts with the reported tissue pattern and lysosomal localisation (HPA: tissue IHC; UniProt O75503 subcellular location). Review counterstain, detection controls, and antibody conditions before assigning it to CLN5 (general IHC practice). |
| Strong staining in skeletal muscle myocytes or lymph node germinal center cells (HPA: Not detected in those cells). | That distribution conflicts with these HPA examples and may reflect cross-reactivity or endogenous detection activity (HPA: tissue IHC; general IHC practice). A negative cell population is a comparison, not proof that every positive cell is specific. |
| Broad, fairly even color over cells and extracellular areas, obscuring cellular boundaries (general IHC practice). | Interpret this as background until detection controls and slide processing are checked (general IHC practice). HPA describes cytoplasmic expression in most tissues, so cytoplasmic staining alone cannot distinguish true signal from widespread background (HPA: tissue IHC). |
| No signal in lung macrophages or duodenal glandular cells (HPA: High in both cell populations). | A blank result in a reported high-staining population warrants a technical check before a negative CLN5 call (HPA: tissue IHC; general IHC practice). HPA's Approved rating has low RNA–staining consistency, so corroborate unexpected biology independently (HPA: reliability). |
💡Expected CLN5 appearanceCall a positive result when cytoplasmic, potentially granular signal is prominent in a reported high-staining population such as lung macrophages or duodenal glandular cells; isolated nuclear or extracellular staining is suspect (HPA: tissue IHC; UniProt O75503 lysosome; general IHC practice).
| Cell and tissue selection (HPA: tissue IHC). | HPA reports High staining in lung macrophages, cerebral cortex glial cells, hippocampal neuronal cells, and several glandular populations; it reports Not detected in skeletal muscle myocytes and selected lymph node cells (HPA: tissue IHC). Score the specified cell population within each tissue. |
| Antibody evidence (HPA: antibody validation; HPA: reliability). | The listed antibody, HPA041788, is Approved for IHC; HPA also notes low consistency between staining and RNA data (HPA: antibody validation; HPA: reliability). Treat an unexpected tissue pattern as provisional and inspect appropriate controls (general IHC practice). |
| Compartment and epitope position (UniProt O75503 topology). | CLN5 spans the membrane at residues 24–40, with residues 41–358 lumenal (UniProt O75503 topology). Epitope position is not supplied, so this record cannot predict which antibody conditions best expose its epitope. |
| Processing and glycosylation (UniProt O75503 processing and glycosylation). | UniProt lists a full-length 1–358 chain, a 44–358 secreted form, and eight glycosylation sites (UniProt O75503). Their effects on the supplied antibody's IHC staining are unreported; do not infer a tissue pattern or retrieval requirement from them. |
| Antigen retrieval (general IHC practice). | Use the IHC-validated antibody's documented retrieval conditions where available, then compare a reported high-staining tissue with detection controls (general IHC practice; HPA: tissue IHC). Neither source here establishes CLN5-specific retrieval or fixation sensitivity. |
| IF/ICC: what pattern can this guide confirm? (HPA: subcellular ICC-IF). | UniProt supports a lysosomal localisation, but HPA supplies no ICC-IF image set or main subcellular location for CLN5 (UniProt O75503 subcellular location; HPA: subcellular ICC-IF). An IF punctate pattern remains a hypothesis for separate validation. |
| Situation | Likely cause | Next action |
|---|---|---|
| A reported high-staining population is blank (HPA: High in lung macrophages or duodenal glandular cells). | Possible assay failure or inadequate signal development (general IHC practice); this result alone does not establish absent CLN5. | Check tissue identity, the detection control, antibody application, and the documented IHC retrieval conditions; repeat with a reported high-staining section (general IHC practice; HPA: tissue IHC). |
| All cells stain strongly, including HPA not-detected populations (HPA: tissue IHC). | Excess background, cross-reactivity, or endogenous detection activity is possible (general IHC practice). | Inspect a no-primary control and detection background; adjust blocking, antibody concentration, or development time as indicated by those controls (general IHC practice). |
| Signal is mainly nuclear (HPA: cytoplasmic expression in most tissues). | The compartment conflicts with reported cytoplasmic staining and lysosomal localisation (HPA: tissue IHC; UniProt O75503 subcellular location). | Confirm the counterstain and cell boundaries, then compare no-primary and reported high-staining tissue controls before scoring (general IHC practice; HPA: tissue IHC). |
| Only extracellular deposits stain (HPA: cytoplasmic expression in most tissues). | Deposits alone do not match HPA's usual cellular pattern; UniProt's secreted form does not establish an extracellular IHC pattern (HPA: tissue IHC; UniProt O75503 processing). | Examine morphology and detection controls; require a supported cellular pattern before interpreting the deposits as CLN5 (general IHC practice; HPA: tissue IHC). |
| Staining differs from an RNA-based expectation (HPA: low RNA–staining consistency). | HPA explicitly reports low consistency between antibody staining and RNA expression (HPA: reliability). | Score the observed cell population and compartment; use independent evidence before claiming that the discrepancy is biological (HPA: tissue IHC and reliability; general IHC practice). |
| An IF image seems to show lysosomal puncta (UniProt O75503 subcellular location). | The appearance is plausible, but HPA provides no CLN5 ICC-IF images or main location to verify it (HPA: subcellular ICC-IF). | Treat the IF assignment as unconfirmed and validate localisation with appropriate controls in the separate IF/ICC workflow (general IF practice). |
Experiment evidence
IHC & IF images showing CLN5 staining
A04893-2 · IHCImmunohistochemistry of paraffin-embedded human appendix using CLN5 antibody at dilution of 1:150 .Perform microwave antigen retrieval with
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000102805-CLN5/tissue/cerebral+cortex#imid_11035451
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000102805-CLN5/tissue/colon#imid_11035369
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000102805-CLN5/tissue/liver#imid_11035362
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000102805-CLN5/tissue/kidney#imid_11035371
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CLN5 IHC & IF
🧪Run caudate first and expect staining in glial cells (HPA: High in caudate glial cells). Use skeletal muscle myocytes as the tissue negative (HPA: Not detected in skeletal muscle myocytes); on the caudate slide, cells without specific staining should retain only the counterstain, but HPA does not identify a particular internal cell population as CLN5-negative (HPA: caudate glial cells High).
Positive control tissue: Caudate (Glial cells, HPA High)
Negative control tissue: Lymph node (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA carries no ICC-IF cell line for CLN5; derive a cell-line control from the positive tissue's cell type (Glial cells) and confirm it by RNA or western blot first.
Technical controls: Include a no-primary, secondary-only control and a host-species- and isotype-matched control for a monoclonal primary, or matched nonimmune host IgG for a polyclonal primary; use identically processed CLN5-knockout material as a biological negative when available (standard IHC practice). Block endogenous peroxidase for chromogenic detection, and distinguish granular brain pigment from chromogen in caudate sections (standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A04893-2 paraffin-section caption does not state a fixative (selected tissue-IHC caption). That caption reports microwave retrieval in 10 mM PBS at pH 7.2 for appendix at 1:150; it does not establish that retrieval condition for caudate (selected tissue-IHC caption). Frozen sections and IF/ICC are not shown to be easier by the supplied evidence; if using IF in brain tissue, assess autofluorescent pigment separately from specific signal (HPA: no ICC-IF images; standard IF practice).
HPA tissue IHC evidence for CLN5
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low consistency between antibody staining and RNA expression data.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.
Positive expression · recommended positive controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Caudate | Glial cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Hippocampus | Neuronal cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Spleen | Cells in red pulp | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CLN5 IHC Tips
CLN5 IHC troubleshooting centers on retrieval, compartment-aware controls, and cautious interpretation of cytoplasmic staining (UniProt O75503 localisation; HPA tissue IHC).
Which retrieval conditions should I try first for CLN5 paraffin sections?
Start with microwave retrieval in 10 mM PBS, pH 7.2, as used for the catalog antibody in paraffin-embedded human appendix (caption A04893-2). The caption gives no microwave duration, so keep the heating program consistent across sections and record its temperature and time before comparing staining (caption A04893-2; standard IHC practice). If staining is weak, compare adjacent sections using citrate pH 6.0 at 95–98 °C for 20 min as the page’s alternate retrieval condition (page retrieval setting). Judge both conditions against tissue morphology and the same positive control, because retrieval can alter signal and background together (standard IHC practice).
How should I assess whether fixation is suppressing CLN5 staining?
The appendix caption describes paraffin sections but does not state the fixative, and target-specific CLN5 fixation sensitivity is unknown (caption A04893-2). Record the actual fixative and fixation interval for each specimen, then compare sections with matched processing and the same retrieval and staining run (standard IHC practice). If signal differs, assess preservation, section thickness, and retrieval consistency before attributing the difference to fixation (standard IHC practice). Include a concurrently stained positive control; high staining in selected HPA cell populations can guide control selection but does not establish any fixation requirement (HPA tissue IHC; standard IHC practice).
What staining pattern fits CLN5 in chromogenic tissue IHC?
Look for intracellular cytoplasmic staining, potentially with a granular distribution, while recognizing that chromogenic IHC alone may not resolve individual lysosomes (HPA tissue IHC; UniProt O75503 localisation; standard IHC practice). CLN5 is assigned to lysosomes and membranes, with residues 24–40 forming a transmembrane segment and residues 41–358 facing the lumen (UniProt O75503 topology). Assess the pattern within identified cell types rather than treating any brown deposit as CLN5 (standard IHC practice). Diffuse extracellular or predominantly nuclear staining needs control-based scrutiny, because neither pattern follows the stated lysosomal and cytoplasmic evidence (UniProt O75503 localisation; HPA tissue IHC).
How can CLN5 processing and epitope position affect paraffin-section staining?
First obtain the antibody’s immunogen or mapped epitope, because the supplied product caption does not identify it (caption A04893-2). CLN5 has one annotated 358-aa chain, a reported 44–358 secreted form, and no annotated isoforms in this record (UniProt O75503 processing and isoforms). An epitope within residues 1–43 would not be present in that reported secreted form; an epitope within residues 44–358 could occur in either form (UniProt O75503 processing). Eight annotated glycosylation sites may matter when interpreting an epitope map, but their effect on this antibody’s staining is unestablished; compare controls under identical retrieval (UniProt O75503 glycosylation; standard IHC practice).
How should I adapt CLN5 localisation checks to multiplex IF?
Use IF as a separate validation experiment: the supplied product image documents paraffin-section IHC, while the HPA subcellular payload lists no ICC/IF images (caption A04893-2; HPA subcellular). Pair CLN5 with a marker that identifies the expected cell population in the specimen, and consider a lysosomal marker when testing compartmental overlap (HPA tissue IHC; UniProt O75503 localisation; standard IF practice). Select spectrally separated fluorophores after checking unstained tissue for autofluorescence, and include single-label controls for bleed-through (standard IF practice). Set permeabilisation according to the mapped epitope: residues 1–23 face the cytoplasm, whereas 41–358 face the lumen, so access across membranes may differ (UniProt O75503 topology; standard IF practice).
What should I change when CLN5 DAB staining looks nonspecific?
Compare a no-primary section with the stained section to identify detection-system deposits before adjusting the catalog antibody (standard IHC practice). In a peroxidase and DAB workflow, apply an endogenous peroxidase block, appropriate protein blocking, and controlled washes as general IHC steps; these are not CLN5-specific validations (standard IHC practice). The appendix image used the antibody at 1:150, which is an evidence-based starting dilution for that specimen and method (caption A04893-2). If background persists, titrate primary concentration and detection development on adjacent sections, preserving a positive control so reduced background is not mistaken for improved specificity (standard IHC practice).
How should I score CLN5 staining across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population, region, and positivity threshold before reviewing outcomes, then apply them consistently to every slide (standard IHC practice). For chromogenic CLN5 IHC, report an H-score from intensity and percentage of positive cells, or report the percentage positive and positive-cell density per mm² when cell counts are reliable (standard IHC practice). Normalize cell-based results to the number of eligible cells in the same compartment and area-based results to analyzable tissue area (standard IHC practice). Score comparable cell types separately, because HPA reports high staining in some glial, glandular, neuronal, macrophage, and fibroblast populations rather than a uniform tissue pattern (HPA tissue IHC).
When is a CLN5-positive IHC signal credible?
Favor reproducible intracellular staining in intact cells that exceeds the no-primary control and fits the expected cytoplasmic or lysosomal distribution (HPA tissue IHC; UniProt O75503 localisation; standard IHC practice). Check cell identity: HPA reports high CLN5 staining in lung macrophages and several other specified populations, while some populations are reported as not detected (HPA tissue IHC). Treat isolated nuclear staining, tissue-edge deposits, necrotic areas, and residual endogenous peroxidase signal as possible artefacts requiring control review (UniProt O75503 localisation; standard IHC practice). HPA labels its tissue IHC “Approved” but reports low consistency with RNA expression, so corroborate consequential differences with an independent assay or antibody when feasible (HPA tissue IHC).
Boster reagents
Best CLN5 / Bis(monoacylglycero)phosphate synthase CLN5 IHC Antibodies
A04893-2 has real IHC data from a human paraffin section (A04893-2 image caption). Human, Mouse, and Rat reactivity is listed; no IF data are shown (catalog reactivity; IF image alts).
Real IHC data
Anti-CLN5 Antibody
Cat # A04893-2
A04893-2 will render with an IHC figure of paraffin-embedded human appendix stained at 1:150 after microwave retrieval in 10 mM PBS, pH 7.2 (A04893-2 image caption). IHC is listed for Human, Mouse, and Rat, but the figure documents human tissue only (catalog applications/reactivity; A04893-2 image caption).
Which to pick: For tissue IHC, choose A04893-2 for paraffin sections; its own caption supports that preparation, but does not report the fixative or establish tissue use (A04893-2 image caption). For IF/ICC, there is no listed IF application or IF figure, and clonality is unreported (catalog applications; IF image alts; catalog clone field). For cross-species IHC, A04893-2 lists Human, Mouse, and Rat reactivity, with pictured IHC evidence limited to human appendix (catalog reactivity; A04893-2 image caption). The selected A04893-2 tissue-IHC caption documents paraffin sections, but does not specify the fixative (selected-SKU IHC image A04893-2).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O75503 (CLN5_HUMAN, Bis(monoacylglycero)phosphate synthase CLN5).
- Human Protein Atlas. CLN5 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. CLN5 subcellular location (ICC-IF): Highest expression in ASC52telo: 45.1 nTPM.
- Human Protein Atlas. CLN5 antibody validation summary (1 antibodies).
- A lysosomal enigma CLN5 and its significance in understanding neuronal ceroid lipofuscinosis. Cellular and molecular life sciences : CMLS 2021 — PMC8195759.
- Efficacy of dual intracerebroventricular and intravitreal CLN5 gene therapy in sheep prompts the first clinical trial to treat CLN5 Batten disease. Frontiers in pharmacology 2023 — PMC10628725.
- Proteolytic processing of the neuronal ceroid lipofuscinosis related lysosomal protein CLN5. Experimental cell research 2015 — PMC4589533.
- Exacerbated neuronal ceroid lipofuscinosis phenotype in Cln1/5 double-knockout mice. Disease models & mechanisms 2013 — PMC3597017.
- PubMed PMID:9662406 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15057823 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CLN5 (Bis(monoacylglycero)phosphate synthase CLN5). Expected localisation Cytoplasmic in tissue (HPA tissue IHC); lysosomal membrane expected (UniProt). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A04893-2 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Caudate (Glial cells); negative Lymph node. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human appendix tissue; fixative not specified (datasheet A04893-2); 2. Fixation: Image fixative and duration unreported (datasheet A04893-2); 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-CLN5, 1:50-1:200 (datasheet A04893-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A04893-2); Caveat: Low staining–RNA concordance complicates interpretation (HPA tissue IHC); Isoform / epitope: No isoforms; cytoplasmic N-terminus absent from 44–358 form (UniProt); Regulation: No specific staining regulator reported (UniProt). Evidence: UniProt O75503, HPA tissue IHC · Approved, HPA cell-line RNA · Highest expression in ASC52telo: 45.1 nTPM, Datasheet A04893-2, PMC10628725. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CLN5/immunohistochemistry-ihc-guide.