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DDX3Y / ATP-dependent RNA helicase DDX3Y · IHC design guide
Design Immunohistochemistry for DDX3Y
Plan DDX3Y chromogenic IHC in paraffin sections using the cytoplasmic tissue pattern reported by HPA (HPA tissue IHC). Interpret staining cautiously because HPA reports that its tissue antibody targets proteins from more than one gene (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
DDX3Y 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 tissue stain; nuclear shuttling is molecular (HPA tissue IHC; UniProt) | |
| Staining pattern | Cytoplasmic staining in germinal center and hematopoietic cells (HPA tissue IHC) | |
| Antigen retrieval | Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen) | |
| Positive control | Tonsil+4 more · see all | |
| Negative control | Cervix+4 more · see all |
Important caveats
Reasons your staining may differ from the expected pattern.
| Fixation | Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific) | |
| Caveat | Antibody may also detect proteins from other genes (HPA tissue IHC) | |
| Regulation | Testis-specific at protein level (UniProt) | |
| Isoform / epitope | Three isoforms; epitope coverage requires checking (UniProt) |
Section 1
Recommended DDX3Y IHC & IF Protocols
The catalog antibody’s IHC-P protocol is accompanied by published DDX3Y methods for primate testis and dysgenetic gonadal sections (PMC9142519; PMC10284372).
| Sample | Paraffin-embedded rat testis tissue; fixative not specified (datasheet A06062-2) |
| Fixation | Image fixative and duration unreported (datasheet A06062-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0, 20 min at 95–98 °C (standard rule: nuclear antigen) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% normal serum of the secondary host, 30 min, room temperature (standard) |
| Primary antibody | Rabbit anti-DDX3Y, 1:50-1:100 (datasheet A06062-2) |
| Primary incubation | Overnight at 4 °C (standard) |
| Detection | HRP-polymer secondary, DAB chromogen 5–10 min (standard) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | DDX3Y-positive staining in germinal center cells of tonsil (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several different tissue types, highly expressed in testis, lymphoid tissue and bone marrow. No signal in the no-primary control. |
💡Decision noteStart with Tris-EDTA pH 9.0 at 95–98 °C for 20 min for the catalog antibody (page retrieval specification). Follow each article’s boric-acid pretreatment when reproducing its DBY-10 method (PMC9142519; PMC10284372).
Section 2
What Is the Expected DDX3Y Staining Pattern?
DDX3Y is a soluble protein that shuttles between cytoplasm and nucleus; it has no transmembrane segment (UniProt O15523). In testis, expect staining predominantly in spermatogonia and occasionally in early spermatocytes (UniProt O15523). HPA reports cytoplasmic IHC staining across several tissues, but its Enhanced tissue profile carries a multi-gene targeting and presumed off-target binding caution (HPA tissue IHC).
| Cytoplasmic staining in spermatogonia, with occasional staining in pre-leptotene or leptotene spermatocytes (UniProt O15523). | This cell distribution fits the reported testis protein pattern (UniProt O15523). Assess localization and cell identity together; intensity alone does not establish DDX3Y specificity (HPA tissue IHC: multi-gene targeting caution). |
| Predominantly extracellular, luminal, or sharply membrane-only staining in testis. | This is a poor match for soluble DDX3Y in IHC (UniProt O15523: cytoplasm, nucleus, no transmembrane segment). Review morphology and staining controls before assigning target expression (general IHC practice). Nuclear signal alone needs context because DDX3Y shuttles (UniProt O15523). |
| Strong staining in an unexpected cell population, such as glandular cells reported as undetected in cervix (HPA tissue IHC). | Consider antibody cross-reactivity or endogenous chromogen-generating activity (HPA tissue IHC: presumed off-target binding; general IHC practice). A stained cell is not automatically DDX3Y-positive; compare with a no-primary control and the tissue pattern. |
| Broad haze across tissue and empty spaces, obscuring cell boundaries. | Treat this as background rather than a localized result (general IHC practice). Check whether the no-primary control also stains, then review blocking, washes, detection chemistry, and the catalog antibody dilution before scoring (general IHC practice). |
| No discernible staining in testis spermatogonia (UniProt O15523). | A negative result in the expected cell population is inconclusive until the run controls pass (UniProt O15523; general IHC practice). Check tissue preservation, retrieval, antibody incubation, and chromogen development against the catalog IHC-P instructions; no DDX3Y-specific fixation effect is established here. |
💡Expected DDX3Y appearanceA convincing IHC positive is cell-associated, chiefly cytoplasmic staining in testis spermatogonia, occasionally in early spermatocytes (UniProt O15523); HPA describes high testis expression but gives no cell-specific testis intensity here (HPA tissue IHC). Diffuse haze or extracellular deposits are suspect (general IHC practice).
| Tissue choice and cell identity | Testis offers a protein-level expectation centered on spermatogonia (UniProt O15523). HPA also reports high staining in tonsil germinal center cells and medium staining in several other cell populations, but its multi-gene antibody caution limits attribution of those signals to DDX3Y (HPA tissue IHC). |
| Antibody specificity and validation | HPA lists IHC status as Enhanced for HPA059583 and Approved for HPA001648 and HPA005631 (HPA antibodies). Enhanced describes reproducibility or orthogonal support for the observed pattern; it does not remove the tissue profile’s presumed off-target binding warning (HPA antibodies; HPA tissue IHC). |
| Compartment and protein topology | Cytoplasmic signal is consistent with HPA tissue IHC; nuclear signal is biologically possible because DDX3Y shuttles (HPA tissue IHC; UniProt O15523). DDX3Y lacks a transmembrane segment, so a membrane-only IHC pattern needs scrutiny (UniProt O15523). |
| Isoforms and antibody epitope | Three isoforms are annotated, but the supplied record gives no epitope mapping or isoform-specific IHC performance (UniProt O15523). Do not infer which isoform a stained cell contains; check the catalog antibody’s stated immunogen and IHC-P validation when interpreting discordant staining (general IHC practice). |
| Endogenous chromogenic activity | Endogenous enzyme activity can produce chromogenic signal independently of the primary antibody (general IHC practice). A no-primary control helps identify detection-derived staining; assess it alongside morphology before calling a weak or widespread signal positive (general IHC practice). |
| IF/ICC Q&A: should its pattern match IHC? | HPA reports mainly cytosolic ICC-IF localization and an additional approved plasma-membrane localization, with a multi-gene antibody caution (HPA subcellular). UniProt also permits nuclear localization through shuttling (UniProt O15523). Use the separate IF/ICC guide for assay design; these observations do not define an IHC-P membrane pattern. |
| Situation | Likely cause | Next action |
|---|---|---|
| Testis section has no spermatogonial signal (UniProt O15523). | The assay may have failed, or the sampled cells may be absent or poorly preserved (general IHC practice). | Confirm spermatogonia on the counterstained section, inspect run controls, and follow the catalog antibody’s IHC-P retrieval and detection instructions (general IHC practice). |
| Tonsil germinal center cells stain strongly (HPA tissue IHC: High). | HPA reports this pattern, but its tissue profile warns of antibodies targeting multiple genes and presumed off-target binding (HPA tissue IHC). | Record the cell type and compartment; compare with testis and appropriate controls. Do not use tonsil staining alone as proof of DDX3Y specificity (HPA tissue IHC; UniProt O15523). |
| Cervical glandular cells stain despite an HPA ‘Not detected’ entry (HPA tissue IHC). | Possible nonspecific antibody binding or endogenous detection activity (HPA tissue IHC: off-target caution; general IHC practice). | Inspect the no-primary control, staining boundaries, and antibody concentration. Treat the result as unresolved until controls support a cell-associated pattern (general IHC practice). |
| Diffuse brown background masks individual cells. | Excess antibody, inadequate blocking or washing, or endogenous detection activity can obscure chromogenic IHC (general IHC practice). | Compare no-primary and tissue controls, then adjust blocking, washing, or antibody dilution within the catalog IHC-P guidance (general IHC practice). |
| Only nuclei stain in an otherwise negative testis section. | DDX3Y can enter nuclei, but an exclusively nuclear pattern lacks the expected cytoplasmic context (UniProt O15523; HPA tissue IHC). | Check that signal follows the expected spermatogonial distribution and that controls are clean before interpreting nuclear staining as DDX3Y (UniProt O15523; general IHC practice). |
| A membrane-only outline dominates the chromogenic section. | DDX3Y has no transmembrane segment; the HPA additional plasma-membrane observation comes from ICC-IF and carries a multi-gene caution (UniProt O15523; HPA subcellular). | Recheck compartment assignment, no-primary staining, and antibody specificity. Seek a cytoplasmic testis pattern before calling the IHC result consistent with DDX3Y (UniProt O15523; HPA tissue IHC). |
Experiment evidence
IHC & IF images showing DDX3Y staining
A06062-2 · IHCImmunohistochemistry of paraffin-embedded rat testis using DDX3Y antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10
A06062-2 · IHCImmunohistochemistry of paraffin-embedded rat brain using DDX3Y antibody at dilution of 1:100 .Perform microwave antigen retrieval with 10 m
A06062-1 · IHCImmunohistochemistry analysis of paraffin-embedded human brain tissue, using DDX3Y Antibody. The picture on the right is blocked with the sy
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000067048-DDX3Y/tissue/cerebral+cortex#imid_566704
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000067048-DDX3Y/tissue/colon#imid_560582
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000067048-DDX3Y/tissue/liver#imid_560575
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000067048-DDX3Y/tissue/kidney#imid_560584
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001648; green = DDX3Y, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA001648; green = DDX3Y, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for DDX3Y IHC & IF
🧪Run tonsil first: germinal center cells should stain (HPA: High in tonsil germinal center cells). Run cervix glandular cells as a negative tissue (HPA: Not detected); on the tonsil slide, assess cells outside the stained germinal center population for background-level signal rather than assuming every neighboring cell is negative (HPA: High in germinal center cells).
Positive control tissue: Tonsil (Germinal center cells, HPA High)
Negative control tissue: Cervix (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show DDX3Y in A-431, U-251MG, U2OS, ASC52telo, RPTEC/TERT1, with annotated localisation: Cytosol (supported) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control and an isotype control matched to the primary antibody’s host species and clonality, alongside a DDX3Y knockout specimen or validated peptide-block control (standard IHC control practice). Block endogenous peroxidase and check for residual chromogen signal in tonsil before scoring (standard chromogenic IHC practice).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported in the supplied evidence, and the selected A06062-2 paraffin-section caption does not report a fixative (selected-SKU tissue-IHC caption). That rat testis IHC caption uses microwave retrieval in 10 mM PBS, pH 7.2, with antibody at 1:100; it does not establish that retrieval is required for tonsil (selected-SKU tissue-IHC caption). The evidence does not establish that frozen sections or IF are easier; in tonsil, assess germinal center staining against background in adjacent cells and the negative controls (HPA: High in tonsil germinal center cells; standard IHC control practice).
HPA tissue IHC evidence for DDX3Y
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 |
|---|---|---|---|---|
| Tonsil | Germinal center cells | High | 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 → |
| Cerebellum | GLUC cells - cytoplasm/membrane | Medium | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cervix | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Endometrium | Cells in endometrial stroma | Not detected | Protein (IHC) | HPA → |
| Heart muscle | Cardiomyocytes | Not detected | Protein (IHC) | HPA → |
| Hippocampus | Glial cells | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced DDX3Y IHC Tips
Troubleshoot DDX3Y chromogenic IHC in paraffin sections by checking retrieval, cell type, compartment, antibody specificity and scoring controls (UniProt O15523; HPA tissue IHC).
Which retrieval conditions should I try first for weak DDX3Y staining?
Start with Tris-EDTA at pH 9.0, 95–98 °C for 20 min (page retrieval rule). If staining remains weak, compare adjacent sections using microwave retrieval in 10 mM PBS, pH 7.2, the condition shown for catalog antibody A06062-2 in paraffin-embedded rat testis (A06062-2 tissue-IHC caption). Keep section thickness, antibody concentration and chromogen development consistent across the comparison (standard IHC practice). The caption used antibody dilution 1:100, but its retrieval result should not be treated as a validated optimum for every specimen or fixation history (A06062-2 tissue-IHC caption; standard IHC practice).
How should I troubleshoot inconsistent staining across differently fixed blocks?
Target-specific DDX3Y sensitivity to fixation is unknown because the selected paraffin-section caption does not report its fixative (A06062-2 tissue-IHC caption). Record each block’s fixative and fixation duration, then compare matched sections with the same retrieval, 1:100 starting dilution and chromogen development (A06062-2 tissue-IHC caption; standard IHC practice). Check morphology alongside signal: damaged tissue or uneven staining can make a fixation comparison uninterpretable (standard IHC practice). If archived blocks vary, optimize retrieval on representative blocks and keep the chosen conditions constant during scoring; do not infer a DDX3Y-specific fixation effect from its localization or modified residues (UniProt O15523; standard IHC practice).
Should DDX3Y staining be nuclear, cytoplasmic or membranous?
Assess cytoplasmic staining first, while recording nuclear staining separately: DDX3Y is annotated in both cytoplasm and nucleus and shuttles between them (UniProt O15523). HPA tissue IHC describes cytoplasmic expression, whereas its subcellular assessment supports cytosol and also reports plasma membrane staining with an antibody cross-target caution (HPA tissue IHC; HPA subcellular). Because DDX3Y has no transmembrane segment, isolated crisp membrane outlining needs independent specificity checks before being called DDX3Y (UniProt O15523 topology; standard IHC practice). Compare compartments in intact cells on serial sections, and avoid interpreting diffuse staining over damaged areas as genuine nuclear redistribution (standard IHC practice).
Could isoforms or epitope masking explain discrepant DDX3Y IHC?
DDX3Y has 3 annotated isoforms, but the supplied antibody caption does not identify its epitope or isoform coverage (UniProt O15523; A06062-2 tissue-IHC caption). First compare retrieval conditions on serial sections before attributing a negative result to isoform expression (standard IHC practice). DDX3Y has helicase ATP-binding residues 209–401, a C-terminal helicase domain at 412–573 and annotated modifications, so epitope placement could matter if later documentation identifies the immunogen (UniProt O15523). Until then, do not claim modification-dependent masking or isoform selectivity; use an independent antibody or orthogonal assay to assess unexpected staining (UniProt O15523; standard IHC practice).
How can I check a surprising IHC pattern by multiplex IF?
For a separate IF experiment, pair DDX3Y with a marker that identifies the cell population being assessed, such as a validated spermatogonial marker when examining testis (UniProt O15523 tissue specificity; standard IF practice). Choose fluorophores after inspecting unstained tissue autofluorescence, and place the weaker channel in a spectral range with less background (standard IF practice). DDX3Y is annotated in cytoplasm and nucleus without a transmembrane segment, so permeabilize fixed cells for intracellular epitopes and titrate detergent to preserve morphology (UniProt O15523 topology and localization; standard IF practice). Compare single-stain and secondary-only controls before treating apparent colocalization as confirmation of the chromogenic IHC pattern (standard IF practice).
What should I check when DDX3Y staining is widespread or diffuse?
Run a no-primary control and inspect staining over edges, folds and damaged regions before changing the antibody dilution (standard IHC practice). For peroxidase-based chromogenic detection, verify the peroxidase block and keep DAB development comparable between sections (standard IHC practice). The catalog caption documents 1:100 in paraffin-embedded rat testis, while HPA reports cytoplasmic staining across several tissues and cautions about presumed off-target binding (A06062-2 tissue-IHC caption; HPA tissue IHC). If diffuse signal persists, titrate antibody and detection strength on matched sections, then require cell-specific staining and an independent specificity check before calling weak widespread color DDX3Y (standard IHC practice; HPA tissue IHC).
How should I score heterogeneous DDX3Y staining in tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring: UniProt reports predominant protein expression in spermatogonia, while HPA describes broader cytoplasmic tissue staining with an off-target caution (UniProt O15523 tissue specificity; HPA tissue IHC). Record the percentage of positive eligible cells and staining intensity, then calculate an H-score from intensity-weighted percentages when that scale fits the study design (standard IHC practice). For spatial counts, report positive cells per mm² of viable, annotated tissue and normalize to the number of eligible cells or sampled tissue area (standard IHC practice). Keep retrieval, counterstain, imaging thresholds and exclusion rules constant across cases (standard IHC practice).
When is an apparent DDX3Y-positive cell credible?
Treat a signal as more credible when it appears in intact cells in a plausible compartment and cell population: DDX3Y is cytoplasmic or nuclear and is reported predominantly in spermatogonia at the protein level (UniProt O15523). HPA’s broader cytoplasmic tissue pattern requires care because its tissue assessment flags presumed off-target binding, and its subcellular assessment uses antibodies targeting multiple genes (HPA tissue IHC; HPA subcellular). Exclude edge staining, necrotic areas and signal reproduced by no-primary controls; check endogenous peroxidase when using DAB (standard IHC practice). Confirm an unexpected cell type or isolated membrane pattern with independent specificity evidence before assigning it to DDX3Y (UniProt O15523 topology; standard IHC practice).
Boster reagents
Best DDX3Y / ATP-dependent RNA helicase DDX3Y IHC Antibodies
Two anti-DDX3Y antibodies have IHC images from paraffin sections of human brain, rat testis and rat brain (catalog IHC captions). Both list IF/ICC; IF images are unreported (catalog applications; image entries).
Real IHC data
Anti-DDX3Y Antibody
Cat # A06062-2
Real IHC data
Anti-DDX3Y Antibody
Cat # A06062-1
A06062-1 lists human and mouse reactivity and IF/ICC, with a peptide-blocked IHC comparison in paraffin-embedded human brain (catalog; A06062-1 IHC caption). A06062-2 lists human, mouse and rat reactivity and IF/ICC, with paraffin-section IHC images from rat testis and rat brain (catalog; A06062-2 IHC captions).
Which to pick: For tissue IHC, choose A06062-1 when the human brain example is most relevant, or A06062-2 for the rat testis or brain examples; the captions identify paraffin sections but do not report the fixative (IHC captions). For IF/ICC, both are listed for those applications, but neither has an IF image in the payload; A06062-1 is identified as polyclonal, while A06062-2 has no reported clonality (catalog). Choose A06062-2 when rat reactivity is needed alongside human and mouse reactivity (catalog reactivity).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry O15523 (DDX3Y_HUMAN, ATP-dependent RNA helicase DDX3Y).
- Human Protein Atlas. DDX3Y tissue IHC expression (reliability: Enhanced).
- Human Protein Atlas. DDX3Y subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the plasma membrane. Caution: Based on antibodies targeting proteins from multiple genes..
- Human Protein Atlas. DDX3Y antibody validation summary (3 antibodies).
- AZFa Y gene, DDX3Y, evolved novel testis transcript variants in primates with proximal 3´UTR polyadenylation for germ cell specific translation. Scientific reports 2022 — PMC9142519.
- Sex chromosome DSD individuals with mosaic 45,X0 and aberrant Y chromosomes in 46,XY cells: distinct gender phenotypes and germ cell tumour risks(§). Systems biology in reproductive medicine 2022 — PMC10284372.
- DDX3Y gene rescue of a Y chromosome AZFa deletion restores germ cell formation and transcriptional programs. Scientific reports 2015 — PMC4601010.
- Construction and analysis of competitive endogenous RNA networks and prognostic models associated with ovarian cancer based on the exoRBase database. PloS one 2024 — PMC11008902.
- PubMed PMID:9381176 — UniProt-cited evidence.
- PubMed PMID:12815422 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for DDX3Y (ATP-dependent RNA helicase DDX3Y). Expected localisation Cytoplasmic tissue stain; nuclear shuttling is molecular (HPA tissue IHC; UniProt). Antigen retrieval: Tris-EDTA pH 9.0 HIER, 95–98 °C, 20 min (rule: nuclear antigen). Validated IHC antibody: A06062-2 at 1:50-1:100 (the product's own IHC validation image is shown). Controls (HPA): positive Tonsil (Germinal center cells); negative Cervix. IHC workflow; specimen preparation unspecified. Protocol steps: 1. Sample: Paraffin-embedded rat testis tissue; fixative not specified (datasheet A06062-2); 2. Fixation: Image fixative and duration unreported (datasheet A06062-2); verify before use.; 3. Sectioning: 4–5 µm sections on charged slides (standard); 4. Deparaffinisation: Xylene, graded ethanol series to water (standard); 5. Antigen retrieval: Heat-induced epitope retrieval in Tris-EDTA buffer, pH 9.0 , 20 min at 95–98 °C…; 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% normal serum of the secondary host, 30 min , room temperature (standard); 8. Primary antibody: Rabbit anti-DDX3Y, 1:50-1:100 (datasheet A06062-2); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across paraffin sections. (standard IHC practice; not target-specific); Caveat: Antibody may also detect proteins from other genes (HPA tissue IHC); Isoform / epitope: Three isoforms; epitope coverage requires checking (UniProt); Regulation: Testis-specific at protein level (UniProt). Evidence: UniProt O15523, HPA tissue IHC · Enhanced, HPA ICC-IF · Mainly localized to the cytosol. In addition localized to the plasma…, Datasheet A06062-2, PMC9142519, PMC10284372. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/DDX3Y/immunohistochemistry-ihc-guide.