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HSPA4 / Heat shock 70 kDa protein 4 · IHC design guide
Design Immunohistochemistry for HSPA4
Plan chromogenic IHC in paraffin sections around the cytoplasmic and nuclear tissue pattern (HPA tissue IHC). Start with the catalog antibody's 2–5 μg/ml IHC range (datasheet A03618-2), and assess ovarian follicle cells and cells in seminiferous ducts as high-staining references while accounting for uncertain HPA reliability (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HSPA4 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 tissue staining (HPA tissue IHC) | |
| Staining pattern | Most tissues show cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A03618-2) | |
| Positive control | Ovary+4 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 (standard IHC practice; not target-specific); its effect on HSPA4 staining is unclear. | |
| Caveat | Staining and RNA show medium consistency; verify locally (HPA tissue IHC) | |
| Regulation | Stress response; induction unreported (UniProt) | |
| Isoform / epitope | 2 isoforms; epitope coverage is unreported (UniProt) |
Section 1
Recommended HSPA4 IHC & IF Protocols
The catalog antibody protocol (datasheet A03618-2) is accompanied by one published chromogenic colorectal IHC protocol (PMC8214019).
| Sample | Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A03618-2) |
| Fixation | Image fixative and duration unreported (datasheet A03618-2); 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 A03618-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A03618-2) |
| Primary antibody | Rabbit anti-HSPA4, 2-5 μg/ml (datasheet A03618-2) |
| Primary incubation | Overnight at 4 °C (datasheet A03618-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet A03618-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HSPA4-positive staining in follicle cells of ovary (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic and nuclear expression in most tissues. No signal in the no-primary control. |
💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03618-2); use citric acid retrieval when reproducing the published protocol (PMC8214019).
Section 2
What Is the Expected HSPA4 Staining Pattern?
HSPA4 is a cytoplasmic protein without a transmembrane segment (UniProt P34932). Tissue IHC shows cytoplasmic and nuclear staining in most tissues, with high staining in ovarian follicle cells and cells in testicular seminiferous ducts (HPA tissue IHC). Treat those examples as useful reference patterns, not definitive controls: HPA rates tissue IHC reliability as Uncertain because antibody staining and RNA expression show only medium consistency (HPA tissue IHC).
| Strong cellular staining in ovarian follicle cells or cells in testicular seminiferous ducts, with cytoplasmic signal and possible nuclear signal (HPA tissue IHC). | This matches the reported high-staining cell populations and broad tissue compartment pattern (HPA tissue IHC). Assess staining in the named cells rather than averaging across the section. A matching appearance supports interpretation but does not establish antibody specificity, because tissue IHC reliability remains Uncertain (HPA tissue IHC). |
| Staining is confined to cell borders, extracellular material, or another location without convincing intracellular staining. | That distribution differs from the reported cytoplasmic and nuclear tissue pattern (HPA tissue IHC), the approved nucleoplasm and cytosol IF locations (HPA subcellular), and the lack of a transmembrane segment (UniProt P34932). Check morphology and controls before assigning it to HSPA4; compartment mismatch alone does not identify its cause. |
| Strong staining appears in adipocytes, cardiomyocytes, smooth muscle cells, or soft-tissue fibroblasts. | Those cell populations were reported as Not detected by tissue IHC (HPA tissue IHC). Recheck cell identity and examine a no-primary control for endogenous detection activity; persistent signal may reflect cross-reactivity. Because the tissue IHC assessment is Uncertain, a single discordant section cannot by itself resolve specificity (HPA tissue IHC). |
| Colour spreads across several cell types, stroma, and areas expected to have little cellular signal. | The reported pattern names specific cells and intracellular compartments, even though expression is widespread (HPA tissue IHC). Broad haze can obscure that distinction. Compare the no-primary control, inspect local tissue background, and reassess antibody concentration and washing as general IHC practice before scoring cells as positive. |
| No convincing staining appears in ovarian follicle cells or cells in testicular seminiferous ducts. | Both were reported as High in tissue IHC (HPA tissue IHC), so first check section quality, detection controls, and assay conditions as general IHC practice. Do not treat absence in one section as proof of biological absence: HPA labels its tissue IHC evidence Uncertain (HPA tissue IHC). |
💡Expected HSPA4 appearanceA plausible positive IHC result shows cytoplasmic staining, sometimes with nuclear staining, strongest in ovarian follicle cells or cells in testicular seminiferous ducts (HPA tissue IHC), while strong isolated membrane or extracellular staining calls for review against the intracellular reference pattern (HPA tissue IHC; UniProt P34932).
| Compartment and topology | UniProt places HSPA4 in the cytoplasm and reports no transmembrane segment (UniProt P34932); HPA tissue IHC also reports nuclear staining, and ICC-IF approves nucleoplasm and cytosol (HPA tissue IHC; HPA subcellular). Interpret nuclear signal alongside these method-specific observations rather than requiring every positive cell to be cytoplasm-only. |
| Cell population and staining level | High staining is reported in ovarian follicle cells and cells in testicular seminiferous ducts; medium staining is reported in several glandular, respiratory epithelial, neuronal, and glial populations (HPA tissue IHC). Adipocytes and cardiomyocytes are reported as Not detected (HPA tissue IHC). Compare like cell populations when reviewing sections. |
| Strength of the IHC evidence | HPA rates the overall tissue IHC assessment Uncertain and describes medium consistency with RNA expression, pending external verification (HPA tissue IHC). Both listed antibodies have Uncertain IHC status, while HPA010023 has Approved ICC status (HPA antibodies). A familiar tissue pattern therefore remains a provisional specificity check, not validation of the catalog antibody. |
| Isoforms and processing | UniProt lists two HSPA4 isoforms and describes the annotated protein chain as residues 1–840, with no signal peptide or propeptide (UniProt P34932). The supplied records give no isoform-specific IHC pattern or antibody epitope, so section staining cannot be assigned to one isoform or explained as cleavage or shedding from these data. |
| Antigen retrieval and detection controls | No target-specific fixation or retrieval effect is supplied by UniProt or HPA (UniProt P34932; HPA tissue IHC). Retrieval optimisation, no-primary controls, and checks for endogenous detection activity are general IHC practice. Interpret changes from those steps as assay behaviour until a controlled comparison establishes their effect in the section being studied. |
| Situation | Likely cause | Next action |
|---|---|---|
| The expected positive cell population is faint or blank. | Possible assay under-detection should be considered before inferring absence; ovarian follicle cells and cells in testicular seminiferous ducts were reported as High, but tissue IHC reliability is Uncertain (HPA tissue IHC). | Confirm the intended cells are present, inspect section and detection controls, then optimise retrieval and antibody concentration within the assay as general IHC practice; record each condition rather than assuming a target-specific fixation effect. |
| Nearly every structure is dark, obscuring cell boundaries. | Excessive assay signal or background may overwhelm the cell-specific pattern reported by HPA (HPA tissue IHC); the appearance alone cannot distinguish antibody binding from endogenous detection activity. | Compare a no-primary control, inspect background outside cells, and adjust detection, washing, or antibody concentration one variable at a time as general IHC practice; score only interpretable cellular staining. |
| Strong staining appears where HPA reports no detection. | Adipocytes, cardiomyocytes, smooth muscle cells, and soft-tissue fibroblasts are listed as Not detected (HPA tissue IHC); cell misidentification, cross-reactivity, or endogenous activity could account for discordance. | Verify morphology and section identity, compare a no-primary control, and seek independent specificity evidence before calling that signal HSPA4; the HPA tissue IHC assessment itself is Uncertain (HPA tissue IHC). |
| The main signal follows membranes or extracellular spaces. | That location conflicts with the intracellular tissue and ICC-IF patterns (HPA tissue IHC; HPA subcellular) and with HSPA4 lacking a transmembrane segment (UniProt P34932). | Review the counterstained morphology and assay controls, then compare another validated section or antibody if available as general IHC practice; avoid interpreting an isolated border pattern as expected HSPA4 staining. |
| Cytoplasmic staining is present, but nuclear staining varies among cells. | UniProt annotates cytoplasm, while HPA reports cytoplasmic and nuclear tissue expression and approves nucleoplasm and cytosol by ICC-IF (UniProt P34932; HPA tissue IHC; HPA subcellular). | Score cytoplasmic and nuclear compartments separately and document which cells show each pattern; variation alone does not invalidate a section, but the Uncertain tissue IHC reliability limits a definitive localisation claim (HPA tissue IHC). |
| IF/ICC question: should nucleoplasmic signal be considered compatible with HSPA4? | HPA lists both nucleoplasm and cytosol as Approved ICC-IF locations, and reports cytoplasmic and nuclear expression in tissue IHC (HPA subcellular; HPA tissue IHC). | Yes: interpret nucleoplasmic signal alongside cytosolic signal and appropriate imaging controls (HPA subcellular; general IF practice). Use the separate IF/ICC guide for method decisions; this IHC interpretation does not establish an IF/ICC protocol. |
Experiment evidence
IHC & IF images showing HSPA4 staining
A03618-2 · IHCIHC analysis of HSPA4 using anti-HSPA4 antibody (A03618-2). HSPA4 was detected in a paraffin-embedded section of human colon cancer tissue.
A03618-2 · IHCIHC analysis of HSPA4 using anti-HSPA4 antibody (A03618-2). HSPA4 was detected in a paraffin-embedded section of mouse ovary tissue. Heat me
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000170606-HSPA4/tissue/cerebral+cortex#imid_2792688
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000170606-HSPA4/tissue/colon#imid_2785438
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000170606-HSPA4/tissue/liver#imid_2785447
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000170606-HSPA4/tissue/kidney#imid_2785456
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010023; green = HSPA4, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA010023; green = HSPA4, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HSPA4 IHC & IF
🧪Run ovary first and expect staining in follicle cells (HPA: High in ovary follicle cells). Use adipose tissue adipocytes as the negative comparator (HPA: Not detected in adipocytes); on the ovary slide, cells outside the identified follicle cells should be assessed for background, but HPA does not establish those cells as target-negative (HPA: ovary follicle-cell row).
Positive control tissue: Ovary (Follicle cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HSPA4 in A-431, U-251MG, U2OS, with annotated localisation: Nucleoplasm (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include no-primary (secondary-only) and rabbit IgG isotype controls matched to the primary antibody’s clonality where known, plus an HSPA4 knockout specimen if available (hero caption: rabbit primary; standard IHC practice). Quench endogenous peroxidase and check for residual chromogen background in the ovary section before scoring follicle cells (hero caption: HRP/DAB detection; standard IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact selected-SKU paraffin-section caption does not state a fixative (A03618-2 caption). The caption reports heat retrieval in EDTA at pH 8.0 for a paraffin section, but does not establish whether retrieval is required for every specimen (A03618-2 caption). Neither an advantage for frozen sections or IF nor an ovary-specific artefact is established by the supplied evidence; assess background in the chosen preparation (HPA: ovary follicle-cell row; A03618-2 caption: paraffin-section IHC).
HPA tissue IHC evidence for HSPA4
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Medium consistency between antibody staining and RNA expression data. Pending external verification.). 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 |
|---|---|---|---|---|
| Ovary | Follicle cells | High | Protein (IHC) | HPA → |
| Testis | Cells in seminiferous ducts | High | Protein (IHC) | HPA → |
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Respiratory epithelial cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Adipose tissue | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HSPA4 IHC Tips
Troubleshoot HSPA4 chromogenic IHC in paraffin sections by checking retrieval, compartment, controls and scoring before comparing specimens.
How should I adjust retrieval if HSPA4 staining is weak in paraffin sections?
Start with heat-mediated antigen retrieval in EDTA pH 8.0 for paraffin sections (datasheet A03618-2). The selected tissue image used 10% goat serum and 2 μg/ml primary antibody overnight at 4°C; use these as matched starting conditions while assessing retrieval (datasheet A03618-2). If staining remains weak, compare adjacent sections using citrate-buffer retrieval as a fallback, recording its pH and heating duration without assuming equivalent epitope exposure (standard IHC practice). Keep section thickness and chromogenic detection consistent, then inspect morphology and a no-primary control before increasing heat exposure further (standard IHC practice).
Can I choose a fixative based on the HSPA4 tissue image?
The selected image documents a paraffin-embedded section but does not state its fixative, so HSPA4-specific fixation sensitivity is unknown (datasheet A03618-2). For a new specimen series, record fixative, fixation duration and tissue thickness, and process comparison samples consistently (standard IHC practice). Compare candidate fixation conditions on adjacent, similarly handled tissue while holding EDTA pH 8.0 retrieval and antibody concentration constant (datasheet A03618-2; standard IHC practice). Judge each condition by preserved morphology, interpretable cellular staining and control sections; neither the tissue staining profile nor protein modifications establish a preferred fixative (HPA tissue IHC; UniProt P34932).
Is nuclear HSPA4 staining plausible when I expect cytoplasmic staining?
Interpret cytoplasmic staining as consistent with the UniProt annotation and assess nuclear staining alongside the reported tissue and cellular observations (UniProt P34932; HPA tissue IHC; HPA subcellular). HPA describes cytoplasmic and nuclear staining in most tissues and lists nucleoplasm and cytosol as approved cellular locations (HPA tissue IHC; HPA subcellular). On paraffin sections, score nuclear and cytoplasmic staining separately within the same defined cell population, using a consistent counterstain and exposure to DAB (standard IHC practice). Check a no-primary control and section edges before treating an isolated nuclear pattern as biological, particularly when tissue morphology is compromised (standard IHC practice).
Could HSPA4 isoforms or modifications explain inconsistent IHC staining?
HSPA4 has 2 annotated isoforms, but the supplied evidence does not map this antibody's epitope to either one (UniProt P34932; datasheet A03618-2). The protein has annotated modified residues, including acetyllysine and phosphoserine, without evidence here that any modification changes this antibody's staining (UniProt P34932). Compare adjacent sections under matched fixation, EDTA pH 8.0 retrieval and primary incubation before attributing a staining difference to an isoform or modification (datasheet A03618-2; standard IHC practice). If that explanation matters experimentally, obtain an epitope map and an independent, isoform-resolving assay; report the IHC result as antibody-defined HSPA4 staining meanwhile (standard IHC practice).
How can IF help check an ambiguous HSPA4 IHC pattern?
Use IF as a separate localisation check when chromogenic HSPA4 staining is difficult to assign to individual cells (standard IHC/IF practice). HPA reports high staining in ovarian follicle cells, so multiplex HSPA4 with a validated follicle-cell marker if those cells are the study population (HPA tissue IHC; standard IF practice). Choose a fluorophore away from the specimen's strongest autofluorescence, and include single-stain and no-primary controls to assess bleed-through and background (standard IF practice). Because HSPA4 is reported in cytosol and nucleoplasm and has no transmembrane segment, assess permeabilisation for access to those compartments; the paraffin IHC caption supplies no IF fixation or antibody validation conditions (HPA subcellular; UniProt P34932; datasheet A03618-2).
How do I separate HSPA4 signal from diffuse DAB background?
The selected image used a rabbit primary, peroxidase-linked detection and DAB development in a paraffin section (datasheet A03618-2). First compare a no-primary section and a reagent control, then check whether unwanted colour follows tissue damage, section edges or the intended cellular compartments (standard IHC practice). Apply an endogenous peroxidase block, verify washing and serum blocking, and develop matched sections for the same DAB interval (standard IHC practice). If diffuse staining persists, titrate the primary around the documented 2 μg/ml condition while retaining EDTA pH 8.0 retrieval; record whether specific cellular contrast improves rather than judging total darkness alone (datasheet A03618-2; standard IHC practice).
What should I score when comparing HSPA4 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region of interest before scoring, because HPA reports differing staining levels across tissue cell types (HPA tissue IHC; standard IHC practice). Within each region, record the percentage of positive cells and an intensity-based H-score from 0–300, scoring nuclear and cytoplasmic compartments separately where discernible (standard IHC practice). Normalise counts to the number of evaluable target cells, or report positive-cell density per mm² of viable tissue when area is the denominator (standard IHC practice). Keep retrieval, staining batch, DAB development and scoring thresholds consistent, and disclose HPA's uncertain tissue-IHC reliability when using its profile for comparison (datasheet A03618-2; HPA tissue IHC; standard IHC practice).
What makes an HSPA4-positive IHC pattern credible rather than artefactual?
A credible pattern shows cellular staining in morphologically intact tissue, with cytoplasmic signal and potentially nuclear signal supported by the reported localisation evidence (UniProt P34932; HPA tissue IHC; HPA subcellular). Judge the stained cell type explicitly: HPA reports high staining in ovarian follicle cells but no detected staining in adipocytes, while assigning its tissue-IHC profile uncertain reliability (HPA tissue IHC). Inspect section edges and necrotic areas, and compare no-primary and peroxidase-block controls before calling diffuse DAB deposits HSPA4-positive (standard IHC practice). Treat staining confined to an unsupported compartment, damaged area or control section as unresolved, and seek independent confirmation before making a biological claim (standard IHC practice).
Boster reagents
Best HSPA4 / Heat shock 70 kDa protein 4 IHC Antibodies
A03618-2 has paraffin-section IHC images from human colon cancer and mouse ovary (image captions). Both antibodies list IF/ICC applications (catalog); neither supplies an IF image (catalog).
Real IHC data
Anti-HSPA4 Antibody ®
Cat # A03618-2
A03618-2 lists IHC and IF/ICC applications and human, mouse, and rat reactivity (catalog); its IHC captions show human colon cancer and mouse ovary paraffin sections (image captions). M03618 lists IHC and IF/ICC applications and human, mouse, and rat reactivity (catalog), with no IHC or IF image supplied (catalog).
Which to pick: For tissue IHC, choose A03618-2: its captions document paraffin-section staining in human colon cancer and mouse ovary, but do not report the fixative (image captions). For human IF/ICC, A03618-2 has a stated 5 μg/ml condition (catalog); M03618 is a rabbit monoclonal alternative, clone IAB-8, with IF/ICC listed but no supplied image (catalog). Both list human, mouse, and rat reactivity (catalog), while the supplied IHC images for A03618-2 cover human and mouse only (image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P34932 (HSP74_HUMAN, Heat shock 70 kDa protein 4).
- Human Protein Atlas. HSPA4 tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. HSPA4 subcellular location (ICC-IF): Localized to the nucleoplasm and cytosol..
- Human Protein Atlas. HSPA4 antibody validation summary (2 antibodies).
- Significant correlation between HSPA4 and prognosis and immune regulation in hepatocellular carcinoma. PeerJ 2021 — PMC8555498.
- Involvement of heat shock protein a4/apg-2 in refractory inflammatory bowel disease. Inflammatory bowel diseases 2015 — PMC4894780.
- HSPA4 Knockdown Retarded Progression and Development of Colorectal Cancer. Cancer management and research 2021 — PMC8214019.
- HSPA4 upregulation induces immune evasion via ALKBH5/CD58 axis in gastric cancer. Journal of experimental & clinical cancer research : CR 2024 — PMC11000359.
- PubMed PMID:15372022 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
- PubMed PMID:8335910 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HSPA4 (Heat shock 70 kDa protein 4). Expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet A03618-2). Validated IHC antibody: A03618-2 at 2-5 μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Ovary (Follicle cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet A03618-2); 2. Fixation: Image fixative and duration unreported (datasheet A03618-2); 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 retrieval: EDTA pH 8.0 (datasheet A03618-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet A03618-2); 8. Primary antibody: Rabbit anti-HSPA4, 2-5 μg/ml (datasheet A03618-2); 9. Primary incubation: Overnight at 4 °C (datasheet A03618-2). Watch for: Fixation: Keep fixation consistent (standard IHC practice; not target-specific); its effect on HSPA4 staining is unclear.; Caveat: Staining and RNA show medium consistency; verify locally (HPA tissue IHC); Isoform / epitope: 2 isoforms; epitope coverage is unreported (UniProt); Regulation: Stress response; induction unreported (UniProt). Evidence: UniProt P34932, HPA tissue IHC · Uncertain, HPA ICC-IF · nucleoplasm and cytosol., Datasheet A03618-2, PMC8214019. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HSPA4/immunohistochemistry-ihc-guide.