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HUWE1 / E3 ubiquitin-protein ligase HUWE1 · IHC design guide
Design Immunohistochemistry for HUWE1
Plan HUWE1 IHC on paraffin sections using the catalog antibody’s 1:50–1:300 IHC range (datasheet A04634-1). Interpret cytoplasmic and nuclear staining (HPA tissue IHC) with consistent fixation and appropriate controls (standard IHC practice).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
HUWE1 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 | Widespread cytoplasmic and nuclear staining (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A04634-1) | |
| Positive control | Adrenal gland+4 more · see all | |
| Negative control | None in HPA (detected in all 45 tissues); use no-primary + isotype controls |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across sections (standard IHC practice; not target-specific) | |
| Caveat | Nuclear staining may predominate in neuronal cells (UniProt) | |
| Regulation | Staining varies by cell type (HPA tissue IHC) | |
| Isoform / epitope | 3 isoforms; verify epitope coverage across variants (UniProt) |
Section 1
Recommended HUWE1 IHC & IF Protocols
The catalog antibody protocol uses Tris-EDTA pH 9.0 retrieval (datasheet A04634-1); the published HUWE1 IHC protocol below uses citrate pH 6.0 retrieval (PMC7329634).
| Sample | Paraffin-embedded human brain tumor tissue; fixative not specified (datasheet A04634-1) |
| Fixation | Image fixative and duration unreported (datasheet A04634-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: Tris-EDTA pH 9.0 (datasheet A04634-1); 20 min, 95–100 °C (standard) |
| 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-HUWE1, 1:50 recommended; image 1:200 (datasheet A04634-1) |
| 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 | HUWE1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: Medium). HPA tissue profile: Ubiquitous cytoplasmic and nuclear expression. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 for the catalog antibody (datasheet A04634-1). If optimizing retrieval, compare the published citrate pH 6.0 method (PMC7329634).
Section 2
What Is the Expected HUWE1 Staining Pattern?
HUWE1 staining is expected in cytoplasm and nuclei across many cell types (HPA tissue IHC: ubiquitous cytoplasmic and nuclear expression). HPA reports medium staining in selected glandular, hematopoietic, basal, and neuronal cells, with low staining in several other cell types (HPA tissue IHC). HUWE1 has no transmembrane segment (UniProt Q7Z6Z7 topology). HPA rates the tissue IHC pattern Approved, with medium consistency between staining and RNA data (HPA tissue IHC).
| Medium cytoplasmic and/or nuclear staining in glandular cells, marrow hematopoietic cells, bronchial basal cells, or brain cells listed as positive (HPA tissue IHC). | This fits the reported tissue pattern (HPA tissue IHC). Compare compartments within the identified cells: UniProt describes HUWE1 as mainly cytoplasmic in most tissues, while nuclear localization in spermatogonia, primary spermatocytes, and neuronal cells is annotated by similarity (UniProt Q7Z6Z7 subcellular location). |
| Staining confined to an unexpected compartment, with no convincing cytoplasmic or nuclear signal in cells expected to stain (HPA tissue IHC). | Treat this as a localisation mismatch, not evidence of a new HUWE1 pattern. HUWE1 is annotated in cytoplasm and nucleus, with mitochondrial recruitment after AMBRA1 interaction (UniProt Q7Z6Z7 subcellular location); the supplied tissue IHC profile does not establish an organelle-specific staining pattern (HPA tissue IHC). |
| Strong staining restricted to cells outside the annotated positive populations, especially when nearby expected cells remain unstained (HPA tissue IHC). | Check cell identification and specificity. The HPA profile is ubiquitous, so absence from its example list alone does not make a cell type wrong; a discordant distribution may reflect cross-reactivity or endogenous chromogen-generating activity (HPA tissue IHC; standard IHC-P practice). |
| Diffuse colour across tissue, extracellular spaces, or the entire section, obscuring cell borders and nuclei. | This is background rather than interpretable cellular localisation (standard IHC-P practice). Assess staining against a no-primary control and tissue morphology before scoring; background cannot be assigned to HUWE1 from the distribution alone (standard IHC-P practice). |
| No convincing staining in a selected HPA medium-staining tissue and cell population (HPA tissue IHC). | Investigate the assay before calling HUWE1 absent. HPA reports medium, rather than uniformly strong, staining in its listed positive populations and rates tissue IHC Approved with medium staining-to-RNA consistency (HPA tissue IHC). Confirm that the relevant cells are present and the detection run worked (standard IHC-P practice). |
💡Expected HUWE1 appearanceCall a section positive when identifiable expected cells show cellular cytoplasmic and/or nuclear staining, often medium in the listed HPA positive populations; diffuse section-wide colour or signal only in discordant cells is suspect (HPA tissue IHC; standard IHC-P practice).
| Tissue and cell selection (HPA tissue IHC) | HPA lists medium staining in several glandular populations, marrow hematopoietic cells, bronchial basal cells, and selected brain cells, but low staining in lung alveolar cells, liver cholangiocytes, and other listed populations. Choose a control by its specified cell population, not its organ name alone (HPA tissue IHC). |
| Compartment and topology (UniProt Q7Z6Z7; HPA tissue IHC) | HUWE1 has no transmembrane segment; UniProt places it in cytoplasm and nucleus and notes conditional mitochondrial recruitment. The HPA tissue IHC summary reports cytoplasmic and nuclear expression. Neither source makes membrane-only tissue staining an expected result (UniProt Q7Z6Z7 topology and subcellular location; HPA tissue IHC). |
| Antibody validation (HPA antibodies; HPA tissue IHC) | HPA002548 and CAB022718 are both Approved for IHC; that status should not be described as Enhanced IHC validation. The tissue profile has medium consistency with RNA expression, so interpret an unexpected compartment or cell distribution alongside controls (HPA antibodies; HPA tissue IHC). |
| Isoforms and processing (UniProt Q7Z6Z7) | UniProt lists 3 isoforms, no signal peptide or propeptide, and one annotated chain spanning residues 1–4374. These facts do not identify the antibody epitope or establish isoform-specific IHC staining; avoid assigning a staining difference to an isoform without separate evidence (UniProt Q7Z6Z7). |
| Situation | Likely cause | Next action |
|---|---|---|
| The selected HPA positive cell population is unstained. | The expected cells may be absent from the examined area, or the IHC-P detection run may have failed; an HPA medium designation does not guarantee strong signal in every section (HPA tissue IHC; standard IHC-P practice). | Verify morphology and cell identity, then inspect a concurrently processed positive control and detection controls. Review the catalog antibody's IHC-P instructions for retrieval and dilution rather than inferring HUWE1-specific settings from HPA staining levels (standard IHC-P practice; HPA tissue IHC). |
| Signal appears only in a compartment that conflicts with the expected cellular pattern. | Non-specific staining or misread morphology is possible; HPA tissue IHC reports cytoplasmic and nuclear expression, while UniProt's mitochondrial recruitment is conditional (HPA tissue IHC; UniProt Q7Z6Z7). | Recheck the counterstain and cell boundaries, compare with no-primary staining, and confirm the pattern in an HPA listed positive cell population before assigning subcellular localisation (standard IHC-P practice; HPA tissue IHC). |
| Unexpected cells stain while the nearby expected population does not. | Cross-reactivity, endogenous detection activity, or cell misidentification may explain a discordant distribution (standard IHC-P practice). HPA's ubiquitous profile prevents treating every unlisted cell as intrinsically negative (HPA tissue IHC). | Identify both populations on the counterstained section, check a no-primary control for detection activity, and compare the cellular pattern with the HPA tissue profile (standard IHC-P practice; HPA tissue IHC). |
| Brown colour spreads through the section and obscures cellular detail. | Background from detection chemistry or nonspecific binding can hide a real cellular pattern (standard IHC-P practice). | Check the no-primary control, blocking and wash steps, and whether chromogen development obscured morphology; then reassess cytoplasmic and nuclear staining in identifiable cells (standard IHC-P practice; HPA tissue IHC). |
| A no-primary section still develops chromogenic signal. | The signal does not require primary-antibody binding and may arise from endogenous activity or the detection system (standard IHC-P practice). | Resolve the control staining with the detection system's recommended blocking and control steps before scoring HUWE1. Do not count matching colour in the test section as target-specific without a clean control (standard IHC-P practice). |
| What pattern should an IF/ICC image show? | HPA supports nucleoplasm and cytosol as the main ICC-IF locations; its additional listed locations have separate support levels (HPA subcellular). Tissue IHC appearance and ICC-IF localisation are distinct observations (HPA tissue IHC; HPA subcellular). | For IF/ICC interpretation, compare cellular signal with nucleoplasm and cytosol and consult the separate IF/ICC guide for that application; do not infer an IF/ICC protocol from the tissue IHC pattern (HPA subcellular; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing HUWE1 staining
A04634-1 · IHCImmunohistochemical analysis of paraffin-embedded human brain tumor. 1, Antibody was diluted at 1:200 (4° overnight). 2, Tris-EDTA, pH9.0 wa
A04634 · IFImmunocytochemistry of ARF-BP1 in Daudi cells with ARF-BP1 antibody at 5 μg/mL.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000086758-HUWE1/tissue/cerebral+cortex#imid_968312
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000086758-HUWE1/tissue/colon#imid_968230
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000086758-HUWE1/tissue/liver#imid_968247
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000086758-HUWE1/tissue/kidney#imid_968232
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB022718; green = HUWE1, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody CAB022718; green = HUWE1, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for HUWE1 IHC & IF
🧪Run bone marrow first and assess staining in hematopoietic cells, which have Medium HUWE1 staining (HPA: bone marrow, hematopoietic cells, Medium). HPA detects HUWE1 in all 45 scored tissues, so there is no validated negative tissue or internal negative cell population; the no-primary and isotype slides should show cells without specific chromogen above background (HPA: no negative tissue rows; standard IHC practice).
Positive control tissue: Adrenal gland (Glandular cells, HPA Medium)
Negative control tissue: None in HPA: HUWE1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show HUWE1 in A-431, U-251MG, U2OS, Sperm, KOLF2.1J, HeLa BAC 3374, HeLa , with annotated localisation: Nucleoplasm (supported), Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only slide and an isotype control matched to the primary antibody’s host species and clonality; use HUWE1 knockout material or a validated immunizing-peptide block as a biological specificity control (standard IHC practice). For bone marrow chromogenic IHC, block endogenous peroxidase and check whether hematopoietic cells retain background staining (HPA: bone marrow, hematopoietic cells; standard IHC practice).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the selected A04634-1 paraffin-section caption does not state its fixative (selected-SKU tissue-IHC caption). That caption reports Tris-EDTA retrieval at pH 9.0 and primary antibody dilution of 1:200 overnight at 4°C; whether HUWE1 detection requires that retrieval condition is unreported (selected-SKU tissue-IHC caption). Whether frozen sections or IF are easier is unreported; in bone marrow, endogenous peroxidase in hematopoietic cells can complicate chromogenic scoring (HPA: bone marrow, hematopoietic cells; standard IHC practice).
HPA tissue IHC evidence for HUWE1
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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 |
|---|---|---|---|---|
| Adrenal gland | Glandular cells | Medium | Protein (IHC) | HPA → |
| Appendix | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bone marrow | Hematopoietic cells | Medium | Protein (IHC) | HPA → |
| Breast | Glandular cells | Medium | Protein (IHC) | HPA → |
| Bronchus | Basal cells | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| None in HPA: HUWE1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead. | ||||
Section 3
Advanced HUWE1 IHC Tips
Troubleshoot HUWE1 staining in paraffin sections by checking retrieval, compartment patterns, controls, and cell-level scoring before interpreting signal (datasheet A04634-1; HPA tissue IHC).
How should I retrieve HUWE1 in paraffin sections when staining is weak?
Use heat-mediated Tris-EDTA at pH 9.0 for HUWE1 antigen retrieval in paraffin sections (datasheet A04634-1). The selected image used this retrieval with primary antibody at 1:200 overnight at 4°C, so reproduce those conditions before changing several variables (caption A04634-1). If staining remains weak, compare a shorter and a longer heating interval on adjacent sections while keeping detection and exposure to chromogen consistent (standard IHC practice). Review tissue morphology and background alongside signal, because excessive heating can damage sections and make apparent gains in staining difficult to interpret (standard IHC practice).
Could fixation explain weak or uneven HUWE1 staining?
The selected paraffin-section caption does not report a fixative, so HUWE1-specific sensitivity to fixation is unknown (caption A04634-1). Record the fixative, fixation duration, specimen thickness, and processing history for each section before attributing a weak result to the antibody (standard IHC practice). Compare similarly processed sections with the same pH 9.0 retrieval and 1:200 primary dilution used in the selected image (caption A04634-1). If signal differs across processing batches, examine morphology and an appropriate control stain first; neither the tissue expression pattern nor HUWE1’s molecular features establishes a fixation effect (standard IHC practice; HPA tissue IHC; UniProt Q7Z6Z7).
Should HUWE1 stain nuclei, cytoplasm, or both in tissue sections?
Expect that cytoplasmic and nuclear staining can both be plausible in paraffin-section IHC: the tissue survey reports ubiquitous expression in those compartments (HPA tissue IHC). The protein record describes mainly cytoplasmic expression in most tissues, with nuclear expression in spermatogonia, primary spermatocytes, and neuronal cells (UniProt Q7Z6Z7). When reviewing neuronal cells in cerebral cortex or caudate, score nuclear and cytoplasmic signal separately instead of collapsing them into one intensity value (HPA tissue IHC; standard IHC practice). A mitochondrial-looking punctate pattern requires independent validation; recruitment to mitochondria after AMBRA1 interaction is a conditional observation, not a universal tissue-staining pattern (UniProt Q7Z6Z7).
Can the antibody distinguish HUWE1 isoforms or a modified epitope?
HUWE1 has 3 listed isoforms, so determine whether the antibody’s documented immunogen lies in a sequence shared by them before assigning staining to one isoform (UniProt Q7Z6Z7; standard IHC practice). The record places its HECT domain at residues 4038–4374 and lists numerous modified residues, including phosphoserines at 648 and 649 (UniProt Q7Z6Z7). Those features do not establish which epitope this antibody detects or whether a modification changes staining (UniProt Q7Z6Z7; standard IHC practice). If an isoform-specific conclusion matters, obtain mapped epitope information and compare an independently validated reagent or orthogonal assay on matched material (standard IHC practice).
How should I check HUWE1 localisation in a separate IF/ICC experiment?
For IF/ICC, pair HUWE1 with a marker identifying the cell population being examined; a neuronal marker is relevant when assessing the nuclear pattern described for neuronal cells (UniProt Q7Z6Z7; standard IF practice). Choose fluorophores after inspecting unstained material for autofluorescence, and include single-label controls before interpreting apparent overlap in a multiplex image (standard IF practice). HUWE1 lacks a transmembrane segment and is reported in cytosol and nucleoplasm, so optimise permeabilisation for access to intracellular epitopes while preserving those compartments (UniProt Q7Z6Z7; HPA subcellular; standard IF practice). Fixation and permeabilisation conditions for this antibody in IF/ICC are not supplied here; validate them within the IF/ICC assay rather than transferring the paraffin-section settings (caption A04634-1; standard IF practice).
How can I reduce diffuse brown signal without losing HUWE1 staining?
First compare a no-primary control with the stained section to separate detection-system background from antibody-dependent signal (standard IHC practice). Block endogenous peroxidase before chromogenic development and control DAB development time, especially when brown precipitate appears outside intact cellular profiles (standard chromogenic IHC practice). The selected image used primary antibody at 1:200 overnight at 4°C and secondary antibody at 1:200 for 45 minutes at room temperature; use these as documented starting conditions (caption A04634-1). Adjust blocking, washes, or antibody concentration one factor at a time, retaining cytoplasmic and nuclear cellular patterns as the readout (HPA tissue IHC; standard IHC practice).
What should I measure when HUWE1 staining varies across a section? ⚠ ANSWER MARKED FOR VERIFICATION
Score intact cells within a defined compartment and tissue region, recording nuclear and cytoplasmic HUWE1 separately because both staining locations are reported (HPA tissue IHC; standard IHC practice). An H-score combines the percentage of cells at each intensity level, whereas percent-positive cells or positive-cell density per mm² answers narrower questions (standard IHC practice). Normalise positive counts to eligible cells, or density to measured viable tissue area, and apply the same threshold and chromogen settings across slides (standard IHC practice). Report region selection and excluded edges or necrosis, since variable tissue composition can change a pooled score without a change in cellular staining (standard IHC practice).
Which HUWE1 staining patterns warrant skepticism before calling a result positive?
Treat staining as more convincing when it resolves into intact-cell cytoplasmic or nuclear patterns, consistent with the tissue survey and protein localisation record (HPA tissue IHC; UniProt Q7Z6Z7). Check cell identity before interpreting a nuclear pattern: neuronal nuclear localisation is described, while the broader tissue survey reports both nuclear and cytoplasmic expression (UniProt Q7Z6Z7; HPA tissue IHC). Signal confined to section edges, necrotic areas, or structures stained in the no-primary control warrants investigation for processing or detection artefact, including endogenous enzyme activity (standard IHC practice). The tissue survey’s reliability is Approved with medium agreement between staining and RNA data, so confirm consequential cell-type or compartment claims independently (HPA tissue IHC; standard IHC practice).
Boster reagents
Best HUWE1 / E3 ubiquitin-protein ligase HUWE1 IHC Antibodies
Two anti-HUWE1 antibodies have image data: paraffin-section IHC in human brain tumor (A04634-1 IHC caption) and IF/ICC in Daudi cells (A04634 image captions). Catalog reactivity includes human and mouse for both, plus rat for A04634 (catalog).
Real IHC data
Anti-HUWE1 Antibody
Cat # A04634-1
Real IF data
Anti-ARF-BP1 HUWE1 Antibody
Cat # A04634
A04634-1 will render with paraffin-section IHC data from human brain tumor; its listed applications are IHC and IF, with human and mouse reactivity (A04634-1 IHC caption; catalog). A04634 will render with Daudi-cell IF/ICC data; its listed applications are IF and ICC, with human, mouse and rat reactivity (A04634 image captions; catalog).
Which to pick: Choose A04634-1 for tissue IHC because its own image documents staining in paraffin-embedded human brain tumor (A04634-1 IHC caption); the fixative is unreported. Choose A04634 for cell IF/ICC because its own images document Daudi-cell staining, including ICC at 5 μg/mL and IF at 20 μg/mL (A04634 image captions). For rat samples, A04634 lists rat reactivity and IF/ICC applications; neither card documents rat staining (catalog; image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry Q7Z6Z7 (HUWE1_HUMAN, E3 ubiquitin-protein ligase HUWE1).
- Human Protein Atlas. HUWE1 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. HUWE1 subcellular location (ICC-IF): Mainly localized to the nucleoplasm and cytosol. In addition localized to the nuclear membrane, mid piece, principal piece and end piece..
- Human Protein Atlas. HUWE1 antibody validation summary (2 antibodies).
- Inverse association between MDM2 and HUWE1 protein expression levels in human breast cancer and liposarcoma. International journal of clinical and experimental pathology 2016 — PMC5782804.
- The E3 ligase HUWE1 inhibition as a therapeutic strategy to target MYC in multiple myeloma. Oncogene 2020 — PMC7329634.
- The E3 ubiquitin ligase HUWE1 is required for KRAS-induced lung cancer. Cell death & disease 2026 — PMC13187489.
- PubMed PMID:15989956 — UniProt-cited evidence.
- PubMed PMID:15989957 — UniProt-cited evidence.
- PubMed PMID:15767685 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for HUWE1 (E3 ubiquitin-protein ligase HUWE1). Expected localisation Cytoplasmic and nuclear tissue staining (HPA tissue IHC). Antigen retrieval: Tris-EDTA pH 9.0 HIER, heat-mediated (datasheet A04634-1). Validated IHC antibody: A04634-1 at 1:50 recommended; image 1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Adrenal gland (Glandular cells); negative None in HPA (detected in all 45 tissues); use no-primary + isotype controls. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded human brain tumor tissue; fixative not specified (datasheet A04634-1); 2. Fixation: Image fixative and duration unreported (datasheet A04634-1); 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: Tris-EDTA pH 9.0 (datasheet A04634-1); 20 min, 95–100 °C (standard); 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-HUWE1, 1:50 recommended; image 1:200 (datasheet A04634-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Nuclear staining may predominate in neuronal cells (UniProt); Isoform / epitope: 3 isoforms; verify epitope coverage across variants (UniProt); Regulation: Staining varies by cell type (HPA tissue IHC). Evidence: UniProt Q7Z6Z7, HPA tissue IHC · Approved, HPA ICC-IF · Mainly localized to the nucleoplasm and cytosol. In addition…, Datasheet A04634-1, PMC7329634. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/HUWE1/immunohistochemistry-ihc-guide.