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- Table of Contents
Plan paraffin-section P4HB IHC around the cytoplasmic staining seen in most tissues (HPA tissue IHC). Use pancreatic exocrine cells as a high-staining reference and start the catalog antibody at 2–5 μg/mL (HPA tissue IHC; datasheet A02335-1).
Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.
| Expected localisation | Cytoplasmic staining in most tissues (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic in most tissues; high in pancreatic exocrine cells (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet A02335-1) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Cerebellum+3 more · see all |
| Fixation | Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific) | |
| Caveat | Surface-associated P4HB may be shed in some cell types (UniProt) | |
| Regulation | No abundance regulator specified (UniProt) | |
| Isoform / epitope | No isoforms listed; signal peptide 1–17 is removed (UniProt) |
Start with the catalog antibody’s IHC-P protocol, then compare the published P4HB staining conditions for kidney, prostate, gastric and bladder tissue (PMC9548632; PMC11304455; PMC6000295; PMC7751343).
| Sample | Paraffin-embedded mouse spleen tissue; fixative not specified (datasheet A02335-1) |
| Fixation | Image fixative and duration unreported (datasheet A02335-1); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet A02335-1); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet A02335-1) |
| Primary antibody | Rabbit anti-P4HB, 2-5μg/ml (datasheet A02335-1) |
| Primary incubation | Overnight at 4 °C (datasheet A02335-1) |
| Detection | Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A02335-1) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | P4HB-positive staining in endocrine cells of appendix (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in most tissues. No signal in the no-primary control. |
P4HB is primarily an endoplasmic reticulum lumen protein with no transmembrane segment (UniProt P07237: location and topology). In paraffin section IHC, expect cytoplasmic staining across many tissues (HPA: cytoplasmic expression in most tissues). Strong reference cells include pancreatic exocrine glandular cells, kidney proximal tubule cell bodies and lung type II alveolar cells (HPA: High). HPA rates the tissue IHC profile Enhanced, reflecting consistency between antibody staining and RNA expression (HPA: Enhanced).
| Strong cytoplasmic staining in pancreatic exocrine glandular cells or kidney proximal tubule cell bodies. | This fits two documented High reference patterns (HPA: pancreas and kidney). Compare staining among the named cells on the same section; the tissue name alone does not establish that every cell should be equally positive. The expected compartment is cytoplasmic in tissue IHC (HPA: tissue profile). |
| Signal appears exclusively nuclear, with little or no cytoplasmic staining in otherwise positive cells. | An exclusively nuclear pattern conflicts with the reported cytoplasmic tissue pattern and ER localization (HPA: tissue profile and subcellular location; UniProt P07237: ER lumen). Treat it as suspect staining and review counterstain, detection background and antibody specificity before scoring it as P4HB. |
| Strong staining predominates in skeletal muscle myocytes or parathyroid glandular cells. | Those specific cell populations are reported Not detected by HPA tissue IHC (HPA: skeletal muscle and parathyroid gland). Unexpected signal may reflect cross-reactivity or endogenous detection activity (general IHC practice). Check a detection-only control; do not assume that every cell in either tissue is a negative reference. |
| Diffuse colour covers cells and spaces without a clear cellular pattern. | This is difficult to reconcile with HPA's cell-associated cytoplasmic profile (HPA: tissue profile). In chromogenic IHC, nonspecific binding, excessive detection signal or residual endogenous activity can obscure localization (general IHC practice). Assess background on a matched control before assigning intensity scores. |
| No convincing signal appears in pancreatic exocrine glandular cells. | HPA reports High staining in that cell population (HPA: pancreas). First verify that those cells are present and recognizable, then review the staining run and controls (general IHC practice). One blank section does not establish biological absence or a P4HB-specific sensitivity to fixation. |
| Compartment and topology | P4HB has an ER lumen assignment and no transmembrane segment (UniProt P07237: location and topology). For chromogenic tissue IHC, interpret a cellular cytoplasmic pattern in context (HPA: tissue profile); topology alone cannot identify the epitope recognized by a particular antibody. |
| Processing and possible surface signal | The precursor has a signal peptide at residues 1–17, with the annotated chain spanning 18–508 (UniProt P07237: processing). Surface association or secretion is described as possible in some cell types (UniProt P07237: location). It does not make widespread membrane-only tissue staining the expected IHC result (HPA: tissue profile). |
| Tissue reference selection | HPA reports High staining in pancreatic exocrine glandular cells and Not detected in skeletal muscle myocytes (HPA: tissue IHC). Use the named cell populations when judging a run; these observations are not a universal positive or negative designation for all cells in those organs. |
| Antibody evidence | Two listed antibodies, HPA018884 and CAB012463, have Enhanced IHC status (HPA: antibody validation). This supports the reported tissue pattern but does not provide an epitope, a working dilution or evidence that a particular antigen retrieval condition is required. |
| IF/ICC Q&A: where is P4HB expected? | The supported main location is the endoplasmic reticulum in the HPA ICC-IF record (HPA: subcellular profile). This localization answers the IF/ICC interpretation question; it does not specify an IF/ICC staining protocol or predict identical image contrast across assays. |
| Situation | Likely cause | Next action |
|---|---|---|
| Known-positive pancreatic cells are blank. | The result conflicts with the reported High pancreatic exocrine cell staining (HPA: pancreas); a failed or weak staining run is possible (general IHC practice). | Confirm the correct cells are in the section, inspect a run control, and review antibody dilution, antigen retrieval and detection steps (general IHC practice). No P4HB-specific retrieval condition is established here. |
| Only nuclei appear positive. | That distribution conflicts with cytoplasmic tissue staining and ER localization (HPA: tissue and subcellular profiles). Counterstain or detection artefact is possible (general IHC practice). | Review the chromogen and counterstain separately, then compare with a control stained using the same detection system (general IHC practice). Score P4HB only where the cellular pattern is credible. |
| Unexpected staining appears in skeletal muscle myocytes. | HPA reports these myocytes as Not detected (HPA: skeletal muscle). Cross-reactivity or endogenous detection activity is possible (general IHC practice). | Check a detection-only control and compare the suspect cells with a documented positive population (general IHC practice; HPA: pancreas). Investigate the unexpected signal before treating it as biological expression. |
| Brown colour is widespread, including between cells. | Diffuse background can hide the cell-associated cytoplasmic pattern reported by HPA (HPA: tissue profile); nonspecific binding or excessive detection signal are possible (general IHC practice). | Compare a matched control, then review blocking, antibody dilution, washing and chromogen development (general IHC practice). Reassess localization after background is controlled. |
| Staining is present but weak in a reference-positive cell population. | A weak result differs from HPA's High category for the named cells (HPA: positive tissue entries). Section and assay performance can also affect apparent intensity (general IHC practice). | Verify cell identity and compare a control from the same run; review dilution, retrieval and detection settings (general IHC practice). Do not infer a P4HB-specific fixation effect from intensity alone. |
| A tissue shows a mixture of strongly stained and unstained cells. | HPA assigns levels to particular cell populations, including High pancreatic exocrine cells and Not detected skeletal muscle myocytes (HPA: tissue IHC). A whole-tissue average can conceal this distinction. | Identify and score the relevant cell types and their cytoplasmic staining separately (HPA: tissue profile; general IHC practice). Compare results with the matching HPA cell-level entry. |
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.
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Appendix | Endocrine cells | High | Protein (IHC) | HPA → |
| Colon | Endocrine cells | High | Protein (IHC) | HPA → |
| Duodenum | Endocrine cells | High | Protein (IHC) | HPA → |
| Epididymis | Glandular cells | High | Protein (IHC) | HPA → |
| Gallbladder | Glandular cells | High | Protein (IHC) | HPA → |
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Cerebellum | Cells in granular layer | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Smooth muscle | Smooth muscle cells | Not detected | Protein (IHC) | HPA → |
Troubleshoot P4HB staining in paraffin sections by checking retrieval, compartment, cell identity and assay controls before comparing signal intensity (datasheet A02335-1; HPA tissue IHC; UniProt P07237).
Catalog anti-P4HB antibodies cover paraffin-section IHC in human, mouse and rat tissues (A02335-1 and M02335-3 image captions), plus cell-based IF in T-47D cells (A02335-1 IF caption).
A02335-1 has paraffin-section IHC images from mouse and rat spleen, human lymphoma and human renal clear cell carcinoma, plus IF in T-47D cells (A02335-1 image captions); M02335-3 has IHC images from human breast carcinoma, human liver and rat liver (M02335-3 image captions). M02335-2 lists IHC and ICC/IF applications, but its supplied figures show IF without an identified sample type (M02335-2 applications and IF captions).
Which to pick: For tissue IHC, choose A02335-1 when the documented paraffin-section workflow fits: its mouse spleen image used EDTA retrieval at pH 8.0 and 2 μg/mL primary antibody; the fixative is unreported (A02335-1 IHC caption). For IF/ICC, A02335-1 has a T-47D IF image at 5 μg/mL, while rabbit monoclonal M02335-2 lists ICC/IF and shows IF at 1:50 without identifying the sample (A02335-1 IF caption; M02335-2 catalog and IF caption). For broader listed species reactivity, choose A02335-1, which lists human, monkey, mouse and rat; its IHC images document human, mouse and rat only (A02335-1 catalog and IHC captions).