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HSPB1 / Heat shock protein beta-1 · IHC design guide
Design Immunohistochemistry for HSPB1
Plan HSPB1 staining in paraffin sections using cytoplasmic squamous epithelial staining as a reference pattern (HPA tissue IHC). Compare intensity within the same cell type, and interpret nuclear signal in light of HSPB1’s reported heat shock response (UniProt).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HSPB1 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 staining in squamous epithelium (HPA tissue IHC) | |
| Staining pattern | Squamous epithelial cells show cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6 HIER, heat-mediated (datasheet M00676-5) | |
| Positive control | Bronchus+4 more · see all | |
| Negative control | Appendix+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). Selected-image fixative and duration unreported (datasheet M00676-5); verify before use. | |
| Caveat | Heat shock can shift HSPB1 into nuclear speckles (UniProt) | |
| Regulation | Abundance changes under stress are unreported (UniProt) | |
| Isoform / epitope | No annotated isoforms or processing; no extracellular segment (UniProt) |
Section 1
Recommended HSPB1 IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M00676-5) is paired with four published HSPB1 tissue-staining protocols (PMC12972742; PMC4502388; PMC5370501; PMC4427334).
| Sample | Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet M00676-5) |
| Fixation | Image fixative and duration unreported (datasheet M00676-5); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Citrate pH 6, 20 min (datasheet M00676-5) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M00676-5) |
| Primary antibody | Mouse monoclonal (clone 3H3) anti-HSPB1, 0.5-1μg/ml (datasheet M00676-5) |
| Primary incubation | Overnight at 4 °C (datasheet M00676-5) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet M00676-5) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | HSPB1-positive staining in respiratory epithelial cells of bronchus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in squamous epithelial cells. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated citrate pH 6 retrieval (datasheet: M00676-5); two published protocols also specify citrate pH 6 (PMC4502388; PMC5370501).
Section 2
What Is the Expected HSPB1 Staining Pattern?
HSPB1 should appear mainly cytoplasmic in squamous epithelial cells, with high staining also reported in cardiomyocytes and several respiratory or glandular cell populations (HPA: tissue IHC, Enhanced reliability). It can enter the nucleus during heat shock and associate with mitotic spindles (UniProt P04792: subcellular location). HSPB1 has no transmembrane segment or signal peptide (UniProt P04792: topology and processing).
| Strong cytoplasmic staining in esophageal squamous epithelial cells, with cell boundaries and the counterstained nuclei still readable. | This is a useful positive pattern: HPA reports high staining in these cells and describes its tissue profile as mainly cytoplasmic in squamous epithelium (HPA: esophagus and tissue IHC profile). Judge intensity against cells on the same section, rather than treating every cell as equally positive (general IHC practice). |
| Predominantly extracellular deposits, or a continuous outline around every cell, with little cytoplasmic staining. | That distribution does not fit HSPB1's usual cytoplasmic location or its lack of a signal peptide and transmembrane segment (UniProt P04792: location, topology and processing). Examine staining controls and tissue morphology before calling it specific (general IHC practice). Nuclear or spindle staining needs separate context because those locations are reported under heat shock or mitosis (UniProt P04792: subcellular location). |
| Strong staining in glial cells of the caudate or germinal center cells of a lymph node. | HPA reports HSPB1 as not detected in those particular cell populations; the label is cell specific, not a claim that the entire tissue is negative (HPA: caudate glial cells; lymph node germinal center cells). Review cell identification and controls for cross-reactivity or endogenous detection activity (general IHC practice). |
| Uniform pale color across cells, extracellular space and the slide background, obscuring cell boundaries. | An indiscriminate deposit cannot support a cell type or compartment call (general IHC interpretation). Compare a matched no-primary control, blocking and wash conditions, and detection reagent performance to distinguish nonspecific staining from a localized HSPB1 pattern (general IHC practice). |
| No signal in esophageal squamous epithelium or heart cardiomyocytes despite preserved tissue morphology. | Both are high-staining populations in HPA tissue IHC, so a blank result warrants a technical check before interpreting the specimen as HSPB1 negative (HPA: esophagus and heart muscle). Check a known-positive control, antibody suitability for IHC-P, retrieval and detection according to the assay instructions (general IHC practice). |
💡Expected HSPB1 appearanceCall an IHC result positive when staining is clearly cytoplasmic and strong in an HPA high population such as esophageal squamous epithelium or heart cardiomyocytes (HPA: tissue IHC); indiscriminate extracellular or all-cell color is suspect (general IHC interpretation).
| Cell type and tissue context | HPA reports high staining in esophageal squamous epithelium and heart cardiomyocytes, but not detected staining in specified caudate glial and lymph node germinal center cells (HPA: tissue IHC). Compare like cell types; a tissue-level label alone can hide mixed populations (general IHC interpretation). |
| Physiological compartment | Interphase HSPB1 is cytoplasmic; it can colocalize with mitotic spindles and move into nuclear speckles during heat shock (UniProt P04792: subcellular location). Do not classify every nuclear signal as an artefact without considering cellular state and controls (general IHC interpretation). |
| Evidence behind the tissue pattern | HPA assigns Enhanced reliability to its tissue IHC profile based on consistency between antibody staining and RNA expression; several listed antibodies have Enhanced IHC validation (HPA: tissue IHC and antibody validation). That evidence supports the reported pattern but does not validate an unspecified catalog antibody (HPA: antibody validation scope). |
| What should IF/ICC show? | HPA's ICC-IF summary reports cytosol and plasma membrane localization, both marked enhanced, with images from A-431, U-251MG and U2OS (HPA: subcellular ICC-IF). UniProt also lists cytoplasm and context-dependent nuclear localization (UniProt P04792: subcellular location). Use the separate IF/ICC guide for its assay workflow; this is an IHC interpretation, not an IF protocol. |
| Chromogenic detection activity | If the assay uses an enzyme-based chromogen, endogenous tissue enzyme activity can contribute color that resembles antibody staining (general IHC practice). A matched no-primary control helps assess this possibility; HPA's HSPB1 tissue levels do not establish whether a particular section has detection-related background (HPA: tissue IHC scope). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected-positive esophageal epithelium is blank. | The observed result conflicts with HPA's high staining report; the cause cannot be inferred from the tissue profile alone (HPA: esophageal squamous epithelial cells). | Run a known-positive section alongside the sample; confirm the antibody is validated for IHC-P, then check retrieval, dilution and detection against its instructions (general IHC practice). Do not infer target-specific fixation sensitivity from HPA. |
| All compartments carry a faint, even chromogen haze. | Poorly localized color can arise from nonspecific binding or detection background (general IHC practice). It cannot establish HSPB1 expression in each cell. | Compare a matched no-primary control; review blocking, wash steps and chromogen development time, then score only a resolved cellular pattern (general IHC practice). |
| Color persists in the no-primary control. | Primary-antibody-independent signal points toward endogenous detection activity or another detection reagent contribution (general IHC practice). | Check the detection chemistry and its appropriate endogenous-activity control or blocking step; repeat the no-primary control before assigning cellular positivity (general IHC practice). |
| A section looks positive only as a surface rim or extracellular deposit. | An extracellular-only pattern conflicts with the reported intracellular localization and lack of a signal peptide or transmembrane segment (UniProt P04792: location, topology and processing). | Inspect morphology and matched controls, and compare with a positive section showing cytoplasmic staining (HPA: tissue IHC profile; general IHC practice). Treat a rim alone as insufficient evidence of specific HSPB1 staining. |
| An HPA not-detected cell population appears strongly positive. | HPA's negative call applies to the named cells, such as caudate glia or lymph node germinal center cells, rather than every cell in those tissues (HPA: tissue IHC). Cell misidentification or nonspecific signal remains possible (general IHC interpretation). | Recheck the cell population on the counterstained section, compare a no-primary control, and assess whether staining is intracellular and reproducible (general IHC practice). |
| Nuclear staining appears in an otherwise interpretable section. | Nuclear translocation can occur during heat shock, so nuclear color alone does not prove artefact (UniProt P04792: subcellular location). HPA's usual tissue IHC profile is mainly cytoplasmic (HPA: tissue IHC). | Check whether cytoplasmic staining and cellular morphology are preserved; compare controls and sample context before scoring nuclear localization as specific (general IHC practice). |
Experiment evidence
IHC & IF images showing HSPB1 staining
M00676-5 · IHCIHC analysis of HSP27 using anti-HSP27 antibody (M00676-5). HSP27 was detected in paraffin-embedded section of human lung cancer tissues. He
M00676-5 · IHCIHC analysis of HSP27 using anti-HSP27 antibody (M00676-5). HSP27 was detected in paraffin-embedded section of human oesophagus squama cance
M00676-5 · IFIF analysis of HSP27 using anti- HSP27 antibody (M00676-5). HSP27 was detected in immunocytochemical section of U20S cell. Enzyme antigen re
PB9237 · IHCIHC analysis of HSP27 using anti-HSP27 antibody (PB9237). HSP27 was detected in paraffin-embedded section of human intestinal cancer tissue.
PB9237 · IHCIHC analysis of HSP27 using anti-HSP27 antibody (PB9237). HSP27 was detected in paraffin-embedded section of human mammary cancer tissue. He
PB9237 · IFIF analysis of HSP27 using anti-HSP27 antibody (PB9237). HSP27 was detected in immunocytochemical section of U20S cells. Enzyme antigen retr
M00676-2 · IHCIHC analysis of HSPB1 using anti-HSPB1 antibody (M00676-2). HSPB1 was detected in a paraffin-embedded section of human colon cancer tissue.
M00676-2 · IHCIHC analysis of HSPB1 using anti-HSPB1 antibody (M00676-2). HSPB1 was detected in a paraffin-embedded section of rat stomach tissue. Heat me
M00676-2 · IFImmunofluorescent analysis using the Antibody at 1:50 dilution.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106211-HSPB1/tissue/cerebral+cortex#imid_12753551
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106211-HSPB1/tissue/colon#imid_12753477
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106211-HSPB1/tissue/liver#imid_12753470
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000106211-HSPB1/tissue/kidney#imid_12753463
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB047330; green = HSPB1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB047330; green = HSPB1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HSPB1 IHC & IF
🧪Run bronchus first: respiratory epithelial cells should stain strongly (HPA: High in bronchus respiratory epithelial cells). Use lymph node germinal center cells as a comparison expected to lack detectable staining (HPA: Not detected in lymph node germinal center cells); interphase epithelial nuclei on the positive slide should be comparatively unstained, while stress can shift HSPB1 into nuclei (UniProt P04792: subcellular location).
Positive control tissue: Bronchus (Respiratory epithelial cells, HPA High)
Negative control tissue: Appendix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HSPB1 in A-431, U-251MG, U2OS, with annotated localisation: Plasma membrane (enhanced), Cytosol (enhanced) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and a host- and class-matched mouse IgG isotype control (caption: mouse primary antibody); use HSPB1-knockout material, or peptide competition if the immunogen is available, as a biological specificity control. For chromogenic detection, block endogenous peroxidase and check endogenous biotin before using the biotin–streptavidin/DAB system (caption: biotinylated secondary, SABC and DAB).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M00676-5 tissue-IHC caption does not state a fixative. Heat retrieval in citrate buffer at pH 6 for 20 minutes is a reported starting condition, but retrieval dependency is unreported (caption: lung cancer paraffin section). Is frozen-section IHC or IF/ICC easier? Frozen-section performance is unreported; ICC-IF images exist for A-431, U-251MG and U2OS (HPA: subcellular images), while tissue IF feasibility and a bronchus-specific artefact are unreported.
HPA tissue IHC evidence for HSPB1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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 |
|---|---|---|---|---|
| Bronchus | Respiratory epithelial cells | High | Protein (IHC) | HPA → |
| Endometrium | Glandular cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Endocrine cells | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Cerebral cortex | Endothelial cells | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Lymph node | Germinal center cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced HSPB1 IHC Tips
Troubleshoot HSPB1 staining in paraffin sections by checking retrieval, cell type and compartment before comparing chromogenic signal across samples.
How should I retrieve HSPB1 in paraffin sections when staining is weak?
Start with heat-mediated retrieval in citrate buffer at pH 6 for 20 minutes (datasheet M00676-5). The documented result used a paraffin section of human lung cancer tissue, but its fixative was not reported, so match your section processing as closely as the available details allow (datasheet M00676-5). Keep heating and cooling conditions consistent across slides, then compare a known positive section with a no-primary control (standard IHC practice). If signal remains weak, adjust retrieval time in a small series while holding antibody concentration and detection constant; excessive retrieval can damage morphology or increase background (standard IHC practice).
Could fixation explain variable HSPB1 staining between paraffin blocks?
Target-specific fixation sensitivity is unknown: the selected HSPB1 caption describes a paraffin section but does not state its fixative (datasheet M00676-5). Record the fixative, fixation duration, block age and processing history for each specimen before attributing a weak result to HSPB1 abundance (standard IHC practice). Compare blocks using the same pH 6 citrate retrieval and the same detection run, with a positive control section on each run (datasheet M00676-5; standard IHC practice). If staining changes with processing history, report that association as a technical limitation; neither HPA tissue patterns nor HSPB1 modifications establish a fixation effect (HPA tissue IHC; UniProt P04792).
Where should HSPB1 staining appear, and when is nuclear signal plausible?
Evaluate cytoplasmic staining first: HSPB1 is cytoplasmic in interphase cells, while nuclear translocation can occur during heat shock (UniProt P04792). Mitotic cells may show spindle association, and nuclear HSPB1 can reside in splicing speckles under stress (UniProt P04792). HPA tissue IHC describes mainly cytoplasmic expression in squamous epithelial cells, whereas its cell imaging reports cytosol and plasma membrane localisation; keep those assay contexts distinct when reading a paraffin section (HPA tissue IHC; HPA subcellular). Score cytoplasmic and nuclear signal separately, and check whether apparent membrane outlines persist in a no-primary control before calling them HSPB1 (standard IHC practice).
How can epitope choice affect interpretation of HSPB1 staining?
The supplied record lists 0 isoforms and one HSPB1 chain spanning residues 1–205, so an isoform-specific staining claim is unsupported here (UniProt P04792). HSPB1 has reported modified residues, including phosphoserines at 15, 26, 65 and 78 (UniProt P04792). Check the catalog antibody's stated immunogen and whether it recognizes total or modified HSPB1 before assigning a phosphorylation state to chromogenic signal (standard IHC practice). If its epitope or modification specificity is unavailable, describe the result as staining with that antibody, and compare matched positive and negative controls rather than interpreting intensity as a particular HSPB1 molecular state (standard IHC practice).
How should I investigate HSPB1 localisation with a complementary IF experiment?
Treat IF as a separate assay: HPA cell imaging places HSPB1 in the cytosol and at the plasma membrane, while tissue IHC mainly reports cytoplasmic staining in squamous epithelial cells (HPA subcellular; HPA tissue IHC). Multiplex HSPB1 with a validated marker for the expected cell type, such as squamous epithelial cells in esophagus, and include single-label controls (HPA tissue IHC; standard IF practice). Choose a fluorophore channel with low tissue autofluorescence and inspect an unstained section before interpreting faint signal (standard IF practice). Because HSPB1 has no transmembrane segment, use controlled permeabilisation when testing intracellular epitopes; document the antibody epitope and compare compartment patterns without treating IF as validation of the paraffin assay (UniProt P04792; standard IF practice).
What should I check when HSPB1 DAB staining is diffuse?
The selected example used 10% goat serum blocking, 1 μg/ml primary antibody overnight at 4°C, a biotinylated secondary and DAB detection (datasheet M00676-5). Run no-primary and secondary-only controls to locate nonspecific detection signal, and include a peroxidase block appropriate to the chromogenic workflow (standard IHC practice). Inspect whether diffuse colour follows section edges, damaged tissue or endogenous pigment rather than cells with interpretable cytoplasm (standard IHC practice). If controls are clean but tissue signal is broad, titrate primary antibody and detection development while retaining a positive control; HSPB1 is reported across many tissues, so widespread staining alone does not prove background (UniProt P04792; standard IHC practice).
How should I compare HSPB1 expression across stained sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA reports high staining in esophageal squamous epithelial cells and cardiomyocytes but no detected staining in several specified cell populations (HPA tissue IHC). For comparable regions, record the percentage of positive cells and an intensity-weighted H-score, with cytoplasmic and nuclear results kept separate (standard IHC practice; UniProt P04792). Normalise counts to evaluable cells in the same annotated compartment, or report positive-cell density per mm² of viable tissue when area is the denominator (standard IHC practice). Use matched section processing, retrieval, imaging and scoring thresholds across groups, and exclude folds, edges and necrosis from the measurement area (standard IHC practice).
How can I distinguish credible HSPB1 staining from artefact?
A credible pattern is cell-associated and predominantly cytoplasmic in interphase cells, with nuclear signal interpreted in light of stress rather than assumed to be constitutive (UniProt P04792). Check cell identity against the tissue context: HPA reports high signal in esophageal squamous epithelial cells and bronchial respiratory epithelial cells, while selected glial and endothelial populations are not detected (HPA tissue IHC). Suspect artefact when colour concentrates at section edges, necrosis or damaged areas, or when the no-primary control reveals endogenous enzyme or detection signal (standard IHC practice). Confirm a disputed pattern across well-preserved regions and controls before assigning biological meaning to weak staining, especially when its compartment or cell type conflicts with the expected pattern (standard IHC practice; HPA tissue IHC).
Boster reagents
Best HSPB1 / Heat shock protein beta-1 IHC Antibodies
Anti-HSPB1 antibodies have IHC images from human cancer tissue and rat stomach sections (catalog IHC captions), plus IF/ICC images from human cells (catalog IF captions).
Real IHC data
Anti-Hsp27/HSPB1 Antibody ® (monoclonal, 3H3)
Cat # M00676-5
Real IHC data
Anti-Hsp27/HSPB1 Antibody ®
Cat # PB9237
Real IHC data
Anti-Hsp27 HSPB1 Rabbit Monoclonal Antibody
Cat # M00676-2
M00676-5 has IHC images from human lung cancer and IF/ICC images from U20S cells; PB9237 has IHC images from human intestinal cancer and IF/ICC images from U20S cells (catalog image captions). M00676-2 has IHC images from human colon cancer and rat stomach sections and lists human, mouse and rat reactivity (catalog image captions; catalog reactivity).
Which to pick: For human paraffin-section IHC, M00676-5 is a mouse monoclonal shown with citrate retrieval, while PB9237 is a rabbit antibody shown with citrate retrieval; both captions use 1 μg/ml primary antibody (catalog antibody details; respective IHC captions). For IF/ICC, either has a cell fluorescence image; choose M00676-5 for a mouse primary or PB9237 for a rabbit primary to match the secondary antibody (catalog IF captions). For cross-species work, M00676-2 lists human, mouse and rat reactivity and has human and rat paraffin-section IHC images with EDTA retrieval; its fixative is unreported (catalog reactivity; M00676-2 IHC captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P04792 (HSPB1_HUMAN, Heat shock protein beta-1).
- Human Protein Atlas. HSPB1 tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. HSPB1 subcellular location (ICC-IF): Localized to the plasma membrane and cytosol..
- Human Protein Atlas. HSPB1 antibody validation summary (5 antibodies).
- Cardiomyocyte-derived HSPB1 regulates TGF-β1 maturation and inhibits endothelial-to-mesenchymal transition in myocardial fibrosis. iScience 2026 — PMC12972742.
- Quantitative proteomic analysis shows differentially expressed HSPB1 in glioblastoma as a discriminating short from long survival factor and NOVA1 as a differentiation factor between low-grade astrocytoma and oligodendroglioma. BMC cancer 2015 — PMC4502388.
- Proteomic Characterization Reveals a Molecular Portrait of Nasopharyngeal Carcinoma Differentiation. Journal of Cancer 2017 — PMC5370501.
- Small Heat Shock Protein Beta-1 (HSPB1) Is Upregulated and Regulates Autophagy and Apoptosis of Renal Tubular Cells in Acute Kidney Injury. PloS one 2015 — PMC4427334.
- PubMed PMID:3714473 — UniProt-cited evidence.
- PubMed PMID:2243808 — UniProt-cited evidence.
- PubMed PMID:10777697 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HSPB1 (Heat shock protein beta-1). Expected localisation Cytoplasmic staining in squamous epithelium (HPA tissue IHC). Antigen retrieval: Citrate pH 6 HIER, heat-mediated (datasheet M00676-5). Validated IHC antibody: M00676-5 at 0.5-1μg/ml (the product's own IHC validation image is shown). Controls (HPA): positive Bronchus (Respiratory epithelial cells); negative Appendix. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung cancer tissues; fixative not specified (datasheet M00676-5); 2. Fixation: Image fixative and duration unreported (datasheet M00676-5); 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: Citrate pH 6 , 20 min (datasheet M00676-5); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M00676-5); 8. Primary antibody: Mouse monoclonal (clone 3H3) anti-HSPB1, 0.5-1μg/ml (datasheet M00676-5); 9. Primary incubation: Overnight at 4 °C (datasheet M00676-5). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific). Selected-image fixative and duration unreported (datasheet M00676-5); verify before use.; Caveat: Heat shock can shift HSPB1 into nuclear speckles (UniProt); Isoform / epitope: No annotated isoforms or processing; no extracellular segment (UniProt); Regulation: Abundance changes under stress are unreported (UniProt). Evidence: UniProt P04792, HPA tissue IHC · Enhanced, HPA ICC-IF · plasma membrane and cytosol., Datasheet M00676-5, PMC12972742, PMC4502388. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HSPB1/immunohistochemistry-ihc-guide.