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HSPA1L / Heat shock 70 kDa protein 1-like · IHC design guide
Design Immunohistochemistry for HSPA1L
Plan paraffin-section HSPA1L IHC around strong staining in late spermatids of testis (HPA tissue IHC). Start with the catalog antibody at 2–5 μg/ml (datasheet A04286-3), and assess staining in light of the reported off-target binding (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HSPA1L 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 and nuclear in spermatids (HPA tissue IHC) | |
| Staining pattern | Late spermatids show cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A04286-3) | |
| Positive control | Testis+3 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 | Off-target staining may resemble HSPA1L signal (HPA tissue IHC) | |
| Regulation | Spermatid expression; no regulator reported (UniProt) | |
| Isoform / epitope | One 1–641 chain; no isoforms annotated (UniProt) |
Section 1
Recommended HSPA1L IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet A04286-3) is accompanied by two published HSPA1L chromogenic IHC protocols (PMC13099889: Immunohistochemistry; PMC7979793: Morphological studies).
| Sample | Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet A04286-3) |
| Fixation | Image fixative and duration unreported (datasheet A04286-3); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A04286-3); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A04286-3) |
| Primary antibody | Rabbit anti-HSPA1L, 2-5 μg/ml (datasheet A04286-3) |
| Primary incubation | Overnight at 4 °C (datasheet A04286-3) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A04286-3) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HSPA1L-positive staining in elongated or late spermatids of testis (HPA tissue IHC: High). HPA tissue profile: Selective cytoplasmic and nuclear expression in spermatids in testis. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet A04286-3); compare 10 mM citrate, pH 6.0, if optimizing retrieval (PMC13099889: Immunohistochemistry).
Section 2
What Is the Expected HSPA1L Staining Pattern?
In paraffin-section IHC, expect selective cytoplasmic and nuclear staining in testicular spermatids, strongest in elongated or late spermatids (HPA: Enhanced tissue IHC; High in elongated or late spermatids). UniProt reports expression in spermatids but does not annotate a subcellular location (UniProt P34931). HSPA1L has no transmembrane segment, signal peptide, or propeptide (UniProt P34931 topology and processing). Interpret the pattern cautiously because HPA reports antibody recognition of proteins from more than one gene (HPA: reliability description).
| Strong cytoplasmic and nuclear staining in elongated or late spermatids, with other testicular cells less conspicuous (HPA: tissue IHC). | This matches the principal testis pattern; score the stained cell population as well as intensity (HPA: High in elongated or late spermatids; general IHC practice). |
| Predominantly membrane-restricted staining, without the expected spermatid cytoplasmic or nuclear pattern (HPA: tissue IHC). | Treat this as discordant with the reference pattern and investigate artefact or nonspecific staining (HPA: selective cytoplasmic and nuclear expression; general IHC practice). UniProt provides no membrane localisation (UniProt P34931). |
| Strong staining in an unexpected cell type while spermatids are weak or unstained (HPA: tissue IHC). | Consider cross-reactivity; also check endogenous chromogenic activity in the detection system (HPA: multiple-gene targeting and presumed off-target binding; general IHC practice). Medium staining in specified ciliated cells can be expected (HPA: bronchus, fallopian tube, nasopharynx). |
| Diffuse chromogen across many cells or tissue spaces, with little cell-specific contrast (general IHC practice). | This does not reproduce HPA's selective spermatid pattern (HPA: tissue IHC). Review blocking, washes, detection background, and counterstain before assigning a biological localisation (general IHC practice). |
| No detectable staining in elongated or late spermatids in a testis section (HPA: expected High staining). | First assess section quality and the IHC run with suitable controls (general IHC practice). An absent positive-control signal cannot establish absence of HSPA1L in the study specimen (HPA: High in elongated or late spermatids; general IHC practice). |
💡Expected HSPA1L appearanceA convincing positive is High, selective cytoplasmic and nuclear staining of elongated or late spermatids; isolated membrane staining or widespread staining of unrelated cells is discordant and needs control review (HPA: testis IHC pattern; general IHC practice).
| Which tissue and cells give the clearest IHC reference? | Testis is RNA-enriched, and elongated or late spermatids stain High (HPA: RNA tissue specificity; tissue IHC). Adipocytes in adipose tissue and adrenal glandular cells are reported as not detected; use any comparator at the stated cell-type level (HPA: tissue IHC). |
| How much confidence does the reference pattern support? | HPA grades tissue IHC Enhanced, with medium agreement between staining and RNA data (HPA: reliability description). Its antibody caution includes recognition of proteins from multiple genes and disregarded presumed off-target staining; morphology alone cannot prove target identity (HPA: reliability description; general IHC practice). |
| Does topology predict a membrane or secreted pattern? | No transmembrane segment, signal peptide, propeptide, or annotated modified residues are reported (UniProt P34931 topology, processing, modified residues). These features do not establish a subcellular location; use the observed IHC pattern for slide interpretation (UniProt P34931; HPA: tissue IHC). |
| What does the separate IF/ICC evidence show? | Q: Where is IF/ICC signal reported? A: Mainly vesicles, flagellar centriole, and annulus; additionally perinuclear theca and calyx (HPA: subcellular ICC-IF). These approved locations carry HPA's multiple-gene antibody caution and do not replace the paraffin IHC reference pattern (HPA: subcellular caution; tissue IHC). |
| Is HSPA1L fixation sensitivity established here? | Target-specific fixation sensitivity is unreported in the supplied UniProt and HPA records (UniProt P34931; HPA: tissue IHC). Document the run's processing conditions when comparing sections; do not infer a HSPA1L fixation effect from staining intensity alone (general IHC practice). |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis control lacks spermatid staining (HPA: expected High staining). | The run, detection reagents, tissue preservation, or antibody conditions may have failed (general IHC practice). | Confirm spermatids are present, inspect counterstain and controls, and review the antibody's IHC validation before interpreting study negatives (HPA: testis IHC; general IHC practice). |
| Signal is widespread and masks cell boundaries (general IHC practice). | Nonspecific reagent binding, inadequate washing, or excessive chromogen development can raise background (general IHC practice). | Compare a no-primary control; review blocking, washes, and detection development, then reassess selective spermatid staining (general IHC practice; HPA: tissue IHC). |
| Chromogen appears in unexpected cells or tissue components (HPA: selective spermatid pattern). | Cross-reactivity is plausible given HPA's multiple-gene and presumed off-target cautions; endogenous detection activity is another general IHC possibility (HPA: reliability description; general IHC practice). | Check a no-primary control and endogenous-activity blocking for the detection system; compare cell identity with HPA's tissue pattern (general IHC practice; HPA: tissue IHC). |
| Only cell borders stain in testis (HPA: cytoplasmic and nuclear pattern). | The compartment pattern conflicts with HPA tissue IHC and is not supported by UniProt topology (HPA: tissue IHC; UniProt P34931 topology). | Check section morphology and background controls; withhold a positive HSPA1L call unless the expected spermatid pattern is also present (general IHC practice; HPA: tissue IHC). |
| Ciliated cells stain outside testis (HPA: tissue IHC). | Medium staining is reported in bronchial and nasopharyngeal ciliated cell bodies and fallopian-tube ciliary rootlets (HPA: tissue IHC). | Check tissue and cell identity before calling this off-target; interpret intensity and compartment alongside the HPA antibody-specificity caution (HPA: tissue IHC; reliability description). |
| Study tissue is negative while the testis control is positive (HPA: testis IHC). | The study cell type may have little detectable staining under the assay conditions; HPA reports several specified cell populations as not detected (HPA: tissue IHC; general IHC practice). | Record the scored cell type and detection threshold, and report the result as not detected in that assay rather than claiming universal absence of protein (general IHC practice). |
Experiment evidence
IHC & IF images showing HSPA1L staining
A04286-3 · IHCIHC analysis of HSPA1L using anti-HSPA1L antibody (A04286-3). HSPA1L was detected in a paraffin-embedded section of human ovarian cancer tis
A04286-3 · IHCIHC analysis of HSPA1L using anti-HSPA1L antibody (A04286-3). HSPA1L was detected in a paraffin-embedded section of human ovarian cancer tis
A04286-3 · IFIF analysis of HSPA1L using anti-HSPA1L antibody (A04286-3). HSPA1L was detected in an immunocytochemical section of HELA cells. Enzyme anti
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000204390-HSPA1L/tissue/cerebral+cortex#imid_12476189
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000204390-HSPA1L/tissue/colon#imid_12476123
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000204390-HSPA1L/tissue/liver#imid_12476124
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000204390-HSPA1L/tissue/kidney#imid_12476117
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA052504; green = HSPA1L, blue = nucleus, red = microtubules.
HPA · ICC-IF CACO-2Human Protein Atlas immunofluorescence, CACO-2, antibody HPA052504; green = HSPA1L, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HSPA1L IHC & IF
🧪Run testis first: elongated or late spermatids should stain (HPA: High in elongated or late spermatids). Use adipose tissue as a negative comparator (HPA: Not detected in adipocytes); on the testis slide, assess other cell types against the no-primary background without assuming they are HSPA1L-negative (HPA: High is assigned specifically to elongated or late spermatids).
Positive control tissue: Testis (Elongated or late spermatids, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HSPA1L in CACO-2, PC-3, U2OS, RPTEC/TERT1, hTERT-RPE1 (serum starved), Sperm, with annotated localisation: Vesicles (approved), Flagellar centriole (approved), Annulus (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and rabbit IgG isotype controls matched to the primary antibody’s clonality, alongside a validated HSPA1L knockout specimen or peptide competition control (catalog caption: rabbit anti-HSPA1L). For chromogenic IHC, quench endogenous peroxidase and check the testis section for background before interpreting DAB staining (catalog caption: peroxidase secondary and DAB detection).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A04286-3 tissue-IHC caption does not state a fixative (catalog caption: fixative not stated). The reported paraffin-section staining used heat-mediated retrieval in EDTA at pH 8.0; this supports testing that retrieval condition but does not establish that retrieval is required (catalog caption: EDTA retrieval). Frozen-section performance and whether IF/ICC is easier are unreported; IF localisation should be interpreted cautiously because the HPA subcellular annotation warns that its antibodies target proteins from multiple genes (HPA subcellular: caution).
HPA tissue IHC evidence for HSPA1L
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Caution, targets protein from more than one gene. Presumed off target binding observed and disregarded.). 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 | Elongated or late spermatids | High | Protein (IHC) | HPA → |
| Bronchus | Ciliated cells (cell body) | Medium | Protein (IHC) | HPA → |
| Fallopian tube | Ciliated cells (ciliary rootlets) | Medium | Protein (IHC) | HPA → |
| Nasopharynx | Ciliated cells (cell body) | 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 → |
| Bone marrow | Hematopoietic cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HSPA1L IHC Tips
Troubleshoot HSPA1L staining by checking retrieval, cellular context and antibody specificity before comparing signal across paraffin sections (datasheet A04286-3; HPA tissue IHC).
Which retrieval conditions should I start with for HSPA1L paraffin IHC?
Start with heat mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04286-3). The demonstrated workflow used antibody A04286-3 at 2 μg/ml overnight at 4°C after retrieval, so keep those conditions consistent while assessing retrieval (datasheet A04286-3). Compare a small series of heating durations on adjacent sections, checking tissue integrity as well as signal; the caption specifies neither duration nor temperature (datasheet A04286-3; standard IHC practice). If staining remains weak, test an alternative retrieval buffer on matched sections as a fallback, with the same detection and controls (standard IHC practice).
How should I troubleshoot weak staining when fixation history is uncertain?
The selected paraffin section caption does not report its fixative, so HSPA1L specific sensitivity to fixation is unknown (datasheet A04286-3). Record each specimen’s fixative and processing history, then compare matched sections processed together under the established EDTA pH 8.0 retrieval condition (datasheet A04286-3; standard IHC practice). If signal varies between blocks, check morphology and a control stain before attributing the difference to HSPA1L abundance (standard IHC practice). Use consistent fixation and processing within a comparison set; neither the HPA staining pattern nor the absence of annotated transmembrane segments establishes a fixation effect (HPA tissue IHC; UniProt P34931 topology; standard IHC practice).
Which staining compartments and cells are plausible in tissue sections?
In testis, expect selective cytoplasmic and nuclear staining in elongated or late spermatids, where HPA reports high expression (HPA tissue IHC). HPA also reports medium staining in ciliated cell bodies of bronchus and nasopharynx and ciliary rootlets of fallopian tube (HPA tissue IHC). Score those cell populations separately from neighboring cells, and inspect nuclear and cytoplasmic signal against matched controls rather than treating every DAB deposit as specific (HPA tissue IHC; standard IHC practice). UniProt gives no subcellular annotation and lists no transmembrane segment, so membrane restricted staining needs independent validation (UniProt P34931 subcellular annotation and topology).
How can I assess epitope specificity when HSPA1L staining is unexpected?
UniProt lists one 1–641 chain and no annotated isoforms, domains, glycosylation sites or modified residues for HSPA1L (UniProt P34931). Those annotations do not identify the catalog antibody’s binding site, so request its immunogen or epitope information before interpreting a retrieval dependent pattern (UniProt P34931; standard IHC practice). HPA warns that its antibodies can recognize proteins from more than one gene and that presumed off target binding was disregarded (HPA tissue IHC). Compare an independently validated antibody or an appropriate loss of target control on matched sections, and require agreement in the expected cells and compartments (standard IHC practice; HPA tissue IHC).
How should I check an HSPA1L pattern using multiplex IF?
On the separate IF/ICC workflow, pair HSPA1L with a validated marker for elongated or late spermatids when examining testis (HPA tissue IHC; standard IF practice). Choose fluorophores after measuring tissue autofluorescence in an unstained section, and place the weaker signal in a channel with lower background (standard IF practice). For an intracellular epitope, include a titrated permeabilisation step; HSPA1L has no annotated transmembrane segment, but the antibody’s exact epitope is unspecified (UniProt P34931 topology; standard IF practice). Include single stain and secondary only controls to assess bleed through and background, and interpret HPA IF locations cautiously because those antibodies target proteins from multiple genes (standard IF practice; HPA subcellular).
What should I check when DAB staining appears widespread?
Check a no primary control and inspect pigment or precipitate before assigning diffuse brown signal to HSPA1L (standard IHC practice). Include an endogenous peroxidase block in the chromogenic workflow, because the selected caption used a peroxidase conjugated secondary antibody and DAB (datasheet A04286-3; standard IHC practice). The demonstrated section received 10% goat serum blocking, 2 μg/ml primary antibody overnight at 4°C, and secondary incubation for 30 minutes at 37°C (datasheet A04286-3). If background persists, titrate primary antibody and shorten chromogen development while retaining matched positive and negative controls (standard IHC practice).
How should I quantify HSPA1L staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and scoring area before reading slides; in testis, score elongated or late spermatids separately from other cells (HPA tissue IHC; standard IHC practice). Record the percentage of positive target cells and intensity, or calculate an H-score using fixed intensity categories and identical thresholds across slides (standard IHC practice). Normalize positive counts to the number of eligible target cells, or report positive cell density per mm² of viable tissue when cell counts are impractical (standard IHC practice). Exclude folds, tissue edges and necrotic regions, and compare sections stained in the same run with the same controls (standard IHC practice).
How do I distinguish convincing HSPA1L signal from artefact?
A convincing testis result enriches in elongated or late spermatids and shows the reported cytoplasmic or nuclear pattern, alongside clean controls (HPA tissue IHC; standard IHC practice). Treat isolated membrane outlining cautiously because UniProt annotates no transmembrane segment and no subcellular location for HSPA1L (UniProt P34931 topology and subcellular annotation). Review staining at tissue edges, in necrotic areas and where endogenous peroxidase or DAB precipitate could create false signal (standard IHC practice). HPA reports medium signals in some ciliated cells but warns about antibodies recognizing multiple genes, so confirm unexpected cell patterns with independent specificity evidence (HPA tissue IHC; standard IHC practice).
Boster reagents
Best HSPA1L / Heat shock 70 kDa protein 1-like IHC Antibodies
The IHC-validated antibody has paraffin-section images from human ovarian cancer and mouse and rat testis, plus IF data from HeLa cells (catalog image captions).
Real IHC data
Anti-HSPA1L Antibody ®
Cat # A04286-3
A04286-3 will render with an IHC image from a paraffin-embedded human ovarian cancer section; its other IHC captions show mouse and rat testis sections (catalog IHC captions). Its IF caption shows staining in HeLa cells (catalog IF caption).
Which to pick: Choose A04286-3 for paraffin-section IHC because its own caption documents that preparation; the fixative is unreported (A04286-3 IHC caption). Choose the same SKU for IF/ICC in human cells because IF and ICC are listed applications and its IF caption shows HeLa cells (catalog applications; A04286-3 IF caption). For cross-species IHC, its listed reactivity covers human, mouse and rat, with paraffin-section images in each species; the catalog lists no clone designation (catalog reactivity; A04286-3 IHC captions; catalog clone field).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P34931 (HS71L_HUMAN, Heat shock 70 kDa protein 1-like).
- Human Protein Atlas. HSPA1L tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HSPA1L subcellular location (ICC-IF): Mainly localized to vesicles, flagellar centriole and annulus. In addition localized to the perinuclear theca and calyx. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. HSPA1L antibody validation summary (2 antibodies).
- Disruption of afferent neural circuits leads to arrhythmia in the animal model of hereditary sensory and autonomic neuropathy 6. Frontiers in neural circuits 2026 — PMC13099889.
- VEGFR3 mitigates hypertensive nephropathy by enhancing mitophagy via regulating crotonylation of HSPA1L. Cell communication and signaling : CCS 2025 — PMC11773936.
- A Vaspin-HSPA1L complex protects proximal tubular cells from organelle stress in diabetic kidney disease. Communications biology 2021 — PMC7979793.
- The regulation of MFG-E8 on the mitophagy in diabetic sarcopenia via the HSPA1L-Parkin pathway and the effect of D-pinitol. Journal of cachexia, sarcopenia and muscle 2024 — PMC11154748.
- PubMed PMID:1700760 — UniProt-cited evidence.
- PubMed PMID:9685725 — UniProt-cited evidence.
- PubMed PMID:14656967 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HSPA1L (Heat shock 70 kDa protein 1-like). Expected localisation Cytoplasmic and nuclear in spermatids (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A04286-3). Validated IHC antibody: A04286-3 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Testis (Elongated or late spermatids); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human ovarian cancer tissue; fixative not specified (datasheet A04286-3); 2. Fixation: Image fixative and duration unreported (datasheet A04286-3); 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 retrieval: EDTA pH 8.0 (datasheet A04286-3); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A04286-3); 8. Primary antibody: Rabbit anti-HSPA1L, 2-5 μg/ml (datasheet A04286-3); 9. Primary incubation: Overnight at 4 °C (datasheet A04286-3). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Off-target staining may resemble HSPA1L signal (HPA tissue IHC); Isoform / epitope: One 1–641 chain; no isoforms annotated (UniProt); Regulation: Spermatid expression; no regulator reported (UniProt). Evidence: UniProt P34931, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to vesicles, flagellar centriole and annulus. In…, Datasheet A04286-3, PMC13099889, PMC7979793. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HSPA1L/immunohistochemistry-ihc-guide.