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RPL35 / Large ribosomal subunit protein uL29 · IHC design guide
Design Immunohistochemistry for RPL35
Plan RPL35 paraffin-section IHC around its general cytoplasmic staining profile (HPA tissue IHC). Colon glandular cells show high staining, while cells in kidney glomeruli have no detected staining and can serve as a comparison (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
RPL35 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 | General cytoplasmic staining (HPA tissue IHC) | |
| Staining pattern | Cytoplasmic staining across many cell types (HPA tissue IHC) | |
| Antigen retrieval | Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen) | |
| Positive control | Appendix+4 more · see all | |
| Negative control | Kidney+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) | |
| Caveat | Staining and RNA show medium consistency (HPA tissue IHC) | |
| Regulation | Low tissue specificity of RNA (HPA tissue RNA) | |
| Isoform / epitope | No annotated isoforms; mature chain spans residues 2–123 (UniProt) |
Section 1
Recommended RPL35 IHC & IF Protocols
Compare the catalog antibody’s IHC-P protocol (catalog datasheet) with a published RPL35 IHC protocol for mouse articular cartilage (PMC11001632).
| Sample | Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A10561-1) |
| Fixation | Image fixative and duration unreported (datasheet A10561-1); 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 citrate buffer, pH 6.0, 20 min at 95–98 °C (standard rule: cytoplasmic / membrane 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-RPL35, 1:50-1:200 (datasheet A10561-1) |
| 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 | RPL35-positive staining in glandular cells of appendix (HPA tissue IHC: High). HPA tissue profile: General cytoplasmic expression. No signal in the no-primary control. |
💡Decision noteStart with citrate pH 6.0 retrieval at 95–98 °C for 20 min (page retrieval rule; UniProt P42766: cytoplasm).
Section 2
What Is the Expected RPL35 Staining Pattern?
RPL35 is a cytoplasmic component of the large ribosomal subunit with no transmembrane segment (UniProt P42766). In paraffin section IHC, expect broadly distributed cytoplasmic staining, including high staining in several glandular, glial and lymphoid cell populations (HPA tissue IHC). HPA rates the tissue staining Approved, with medium consistency between antibody staining and RNA expression (HPA tissue IHC).
| Cytoplasmic staining in appendix, colon or duodenal glandular cells, or cerebral cortex glial cells. | These are reported High populations and fit RPL35's cytoplasmic localisation (HPA tissue IHC; UniProt P42766). Compare intensity within the same staining run; the HPA levels describe reference observations, not a required score for every specimen (HPA tissue IHC). |
| Predominantly nuclear, membranous or extracellular staining, with little cytoplasmic signal. | That distribution conflicts with the reported cytoplasmic tissue profile and UniProt localisation (HPA tissue IHC; UniProt P42766). Check localisation against a known positive section and a primary antibody omission control before interpreting it as RPL35 (general IHC practice). |
| Strong staining in kidney glomerular cells, liver cholangiocytes or skeletal muscle myocytes. | HPA lists these specific cell populations as Not detected, so prominent signal needs scrutiny; that designation does not mean every cell in the named tissue is negative (HPA tissue IHC). Check cell identification, antibody dependent staining and endogenous detection activity (general IHC practice). |
| Diffuse colour across cells and tissue spaces, obscuring cell boundaries. | A widespread haze cannot be scored as the reported cytoplasmic cell staining (HPA tissue IHC). Review a primary antibody omission control, chromogen development and washing to locate the background source (general IHC practice). |
| No cytoplasmic signal in an otherwise interpretable appendix glandular or cerebral cortex glial section. | Those populations are reported High, making them useful reference checks, although HPA reports only medium staining–RNA consistency (HPA tissue IHC). Confirm tissue preservation and the staining run with appropriate controls before calling the sample RPL35 negative (general IHC practice). |
💡Expected RPL35 appearanceA credible positive result is cell associated cytoplasmic chromogen, potentially strong in HPA High glandular or glial populations; dominant nuclear or tissue wide diffuse colour is discordant with the reported pattern (HPA tissue IHC; UniProt P42766).
| Cell population within a tissue | Use the named population when comparing slides: appendix glandular cells are High, while glomerular cells in kidney are Not detected (HPA tissue IHC). A tissue name alone does not define the expected result for all its cells (HPA tissue IHC). |
| Strength of the tissue reference | The tissue IHC profile is Approved, with medium consistency against RNA expression (HPA tissue IHC). Treat a mismatch as a reason to check controls and morphology, rather than as proof of either a biological change or an assay failure (HPA tissue IHC; general IHC practice). |
| Target location and processing | UniProt places RPL35 in the cytoplasm and records no transmembrane segment or signal peptide; its annotated chain spans residues 2–123 (UniProt P42766). These annotations support a cellular cytoplasmic interpretation, but do not specify an IHC epitope or retrieval condition (UniProt P42766). |
| IF/ICC comparison | What should IF/ICC show? HPA reports approved endoplasmic reticulum and cytosol localisation in ICC-IF (HPA subcellular). That finer pattern can inform interpretation, but it does not establish that both compartments will resolve separately in chromogenic paraffin section IHC (HPA subcellular; general IHC practice). |
| Antibody evidence | HPA lists one rabbit polyclonal antibody, HPA006047, with Approved IHC and Approved ICC status (HPA antibodies). The supplied record does not show an Enhanced IHC designation or an independent antibody comparison (HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| Expected High cell population has no visible cytoplasmic staining. | The run may lack detectable signal; an absent result alone cannot distinguish assay performance from specimen variation (HPA tissue IHC; general IHC practice). | Stain a reference section containing HPA High cells in the same run, then review antibody dilution, antigen retrieval and detection controls as general IHC checks; no RPL35 specific retrieval condition is supplied (HPA tissue IHC; general IHC practice). |
| Signal appears mainly nuclear or along cell surfaces. | The compartment conflicts with RPL35's cytoplasmic annotation and the general cytoplasmic tissue profile (UniProt P42766; HPA tissue IHC). | Recheck morphology and compare with a reference positive section; use a primary antibody omission control to assess detection related staining (general IHC practice). |
| HPA Not detected cell populations appear strongly positive. | Wrong cell identification, antibody dependent staining or endogenous detection activity may explain the discrepancy; the slide alone cannot choose among them (HPA tissue IHC; general IHC practice). | Identify the stained cells on the counterstained section, then compare omission and detection controls; interpret the named cell population, not the whole tissue, against HPA (HPA tissue IHC; general IHC practice). |
| Diffuse chromogen prevents cytoplasmic scoring. | Background from detection chemistry or insufficient washing can obscure a cell associated pattern (general IHC practice). | Review the omission control and reagent blank, check washing and chromogen development, and rescore only where cell boundaries and cytoplasm remain distinguishable (general IHC practice). |
| Uneven intensity across regions of the same section. | Regional tissue condition or staining variation can change apparent intensity; HPA's cell level reference does not establish the cause in this specimen (HPA tissue IHC; general IHC practice). | Check section integrity, staining uniformity and same run controls before comparing regions or assigning an intensity score (general IHC practice). |
| IF/ICC shows endoplasmic reticulum and cytosol detail that is unclear in IHC. | HPA's approved endoplasmic reticulum and cytosol call comes from ICC-IF; the tissue IHC profile is reported as general cytoplasmic staining (HPA subcellular; HPA tissue IHC). | Score chromogenic IHC as cytoplasmic where supported by cell morphology, and use the separate IF/ICC guide for fluorescence localisation decisions (HPA tissue IHC; HPA subcellular). |
Experiment evidence
IHC & IF images showing RPL35 staining
A10561-1 · IHCImmunohistochemistry (IHC) analyzes of RPL35 (M91) pAb in paraffin-embedded human lung carcinoma tissue at 1:100.
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000136942-RPL35/tissue/cerebral+cortex#imid_2059400
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000136942-RPL35/tissue/colon#imid_2059326
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000136942-RPL35/tissue/liver#imid_2059327
HPA · PancreasHuman Protein Atlas IHC, Pancreas. Source: https://www.proteinatlas.org/ENSG00000136942-RPL35/tissue/pancreas#imid_2059278
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006047; green = RPL35, blue = nucleus, red = microtubules.
HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006047; green = RPL35, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for RPL35 IHC & IF
🧪Run colon first; its glandular cells should stain (HPA: High in colon glandular cells), and use kidney glomerular cells as the negative comparator (HPA: Not detected in kidney glomerular cells). On the colon slide, assess stromal cells for background staining, but do not designate them RPL35-negative without validation because the supplied HPA row reports only glandular cells.
Positive control tissue: Appendix (Glandular cells, HPA High)
Negative control tissue: Kidney (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RPL35 in A-431, U-251MG, U2OS, with annotated localisation: Endoplasmic reticulum (approved), Cytosol (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only control; a host-species-matched nonimmune polyclonal IgG control; and RPL35-knockout material as a biological negative (catalog antibody: pAb). For colon chromogenic IHC, quench endogenous peroxidase and inspect inflammatory cells for residual signal; block endogenous biotin if using avidin–biotin detection.
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected 1:100 paraffin-section lung carcinoma caption does not state its fixative (SKU A10561-1 caption: fixative unreported). Antigen-retrieval dependency is unreported; optimize retrieval on the positive and negative tissues before scoring. Frozen sections or IF cannot be judged easier from the supplied evidence, although ICC-IF images exist for A-431, U-251MG and U2OS (HPA: subcellular); in colon, distinguish glandular signal from mucin or inflammatory-cell background.
HPA tissue IHC evidence for RPL35
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 |
|---|---|---|---|---|
| Appendix | Glandular cells | High | Protein (IHC) | HPA → |
| Breast | Glandular cells | High | Protein (IHC) | HPA → |
| Cerebral cortex | Glial cells | High | Protein (IHC) | HPA → |
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Duodenum | Glandular cells | High | Protein (IHC) | HPA → |
Undetected expression · recommended negative controls
| Tissue | Cell type | Level | Evidence | Source |
|---|---|---|---|---|
| Kidney | Cells in glomeruli | Not detected | Protein (IHC) | HPA → |
| Liver | Cholangiocytes | Not detected | Protein (IHC) | HPA → |
| Parathyroid gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Skeletal muscle | Myocytes | Not detected | Protein (IHC) | HPA → |
| Soft tissue | Fibroblasts | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced RPL35 IHC Tips
Troubleshoot RPL35 chromogenic IHC in paraffin sections using the stated retrieval conditions, documented staining patterns, and matched controls.
How should I adjust retrieval when RPL35 staining is weak or uneven?
For RPL35 paraffin IHC, begin with citrate pH 6.0 heat-induced epitope retrieval at 95–98 °C for 20 min (page retrieval specification). Compare adjacent sections processed together, checking that the temperature is reached throughout the vessel and that sections remain covered by buffer (standard IHC practice). If staining remains weak, vary retrieval time on adjacent sections while keeping citrate pH 6.0, the antibody dilution, and detection conditions constant; score both signal and tissue damage (standard IHC practice). Judge improvement by reproducible cytoplasmic staining in viable cells, since RPL35 is cytoplasmic (UniProt P42766) and the tissue profile describes general cytoplasmic expression (HPA tissue IHC).
Could fixation explain variable RPL35 staining between paraffin blocks?
The selected antibody caption documents staining in paraffin-embedded human lung carcinoma at 1:100, but does not report its fixative (A10561-1 tissue-IHC caption). Target-specific sensitivity to fixative choice or fixation duration is therefore unknown from the supplied evidence (A10561-1 tissue-IHC caption). For a controlled comparison, document cold ischemia, fixative, fixation duration, processing schedule, section age, and retrieval conditions for each block, then stain sections in the same run (standard IHC practice). If blocks differ, compare viable areas and a matched control before attributing intensity differences to RPL35 biology; fixation and processing can alter antigen accessibility in paraffin IHC (standard IHC practice).
Where should RPL35 signal appear, and how should I assess a nuclear pattern?
Expect predominantly cytoplasmic chromogenic staining: UniProt places RPL35 in the cytoplasm, and the tissue-IHC profile reports general cytoplasmic expression (UniProt P42766; HPA tissue IHC). Cell imaging identifies cytosol and endoplasmic reticulum as approved locations, although a chromogenic paraffin section may not resolve those pools separately (HPA subcellular; standard IHC practice). RPL35 has no annotated transmembrane segment, so a sharply outlined plasma membrane pattern alone needs further scrutiny (UniProt P42766 topology). If staining is mainly nuclear, compare adjacent viable cells, a no-primary control, and an independently validated antibody before calling the pattern RPL35-specific (standard IHC practice).
Could an isoform or modified epitope account for inconsistent staining?
The supplied record lists 0 isoforms and a mature chain spanning residues 2–123, so no annotated isoform switch explains a changing IHC pattern (UniProt P42766). It also lists acetylated lysines at 19 and 43 and phosphoserine at 29, but the antibody epitope is not supplied (UniProt P42766; supplied antibody evidence). Do not assign staining loss to those modifications without epitope mapping and direct comparison of modified and unmodified antigen (standard antibody validation practice). First compare section processing and retrieval, then test a second antibody recognizing a documented distinct epitope on matched sections if epitope-specific interference remains a concern (standard IHC practice).
How can IF help check the cell type and compartment seen by IHC?
Use IF/ICC as a complementary localisation check: cell imaging places RPL35 in the cytosol and endoplasmic reticulum, while tissue IHC describes general cytoplasmic expression (HPA subcellular; HPA tissue IHC). Multiplex RPL35 with a cell marker appropriate to the sampled tissue, such as cytokeratin when evaluating glandular epithelium, and include single-label controls (HPA tissue IHC; standard IF practice). Choose a far-red fluorophore and inspect an unstained section when tissue autofluorescence could obscure signal (standard IF practice). Because RPL35 lacks a transmembrane segment and its antibody epitope is unmapped, use controlled permeabilisation to access intracellular antigen, then compare staining with and without permeabilisation before assigning an endoplasmic reticulum-facing epitope (UniProt P42766 topology; supplied antibody evidence; standard IF practice).
What should I change when diffuse DAB obscures RPL35 staining?
Start with a no-primary control and examine whether diffuse color persists, particularly near damaged tissue, blood, or section edges (standard chromogenic IHC practice). Use a peroxidase block before horseradish peroxidase detection, a protein block, adequate washes, and a controlled DAB development time; these are general workflow controls, not RPL35-specific validation (standard chromogenic IHC practice). The selected caption reports paraffin-section staining at 1:100, so treat that dilution as a documented starting point for the exact catalog antibody rather than an optimum for every block (A10561-1 tissue-IHC caption). Titrate primary antibody and development time separately while retaining a positive reference section and scoring whether cytoplasmic signal remains distinguishable from background (HPA tissue IHC; standard IHC practice).
How should I score RPL35 across sections with different cell composition? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and viable tissue area before scoring, and count cytoplasmic staining in that population because RPL35 has a reported cytoplasmic tissue pattern (HPA tissue IHC; standard IHC practice). An H-score combines percentages of cells at intensity 0–3 into a 0–300 score; also report the percentage of positive target cells and the number assessed (standard IHC scoring practice). If cell abundance itself matters, report positive cells per mm² of viable tissue separately from staining intensity (standard image-analysis practice). Keep exposure, DAB development, thresholds, and reference controls consistent, and normalise cell-based results to the eligible cell count rather than the entire mixed-cell section (standard IHC practice).
How can I distinguish genuine RPL35 staining from section artefact?
A credible result is reproducible in viable-cell cytoplasm, consistent with UniProt cytoplasmic localisation and the general cytoplasmic tissue-IHC profile (UniProt P42766; HPA tissue IHC). Use the relevant cell population: high staining is reported in appendix glandular cells, whereas staining is not detected in kidney glomerular cells, so whole-tissue labels alone are inadequate comparators (HPA tissue IHC). Treat isolated nuclear or plasma membrane outlines, edge-only color, and staining confined to necrosis as reasons to repeat controls and inspect morphology before assigning specificity (UniProt P42766 topology; standard IHC practice). Check a no-primary slide for endogenous detection signal, and interpret discordance cautiously because the tissue-IHC assessment reports medium consistency between antibody staining and RNA expression (standard IHC practice; HPA tissue IHC).
Boster reagents
Best RPL35 / Large ribosomal subunit protein uL29 IHC Antibodies
One anti-RPL35 antibody has real IHC data from paraffin-embedded human lung carcinoma (A10561-1 image caption). Mouse and rat reactivity is listed; IF data is absent (catalog reactivity/images).
Real IHC data
Anti-RPL35 (M91) Antibody
Cat # A10561-1
A10561-1 lists IHC and human, mouse, and rat reactivity (catalog applications/reactivity). Its IHC image shows paraffin-embedded human lung carcinoma stained at 1:100 (A10561-1 image caption).
Which to pick: Choose A10561-1, described as a rabbit pAb, for paraffin-section IHC; its image shows human lung carcinoma at 1:100, and its listed IHC range is 1:50–1:200 (catalog host; A10561-1 image caption; datasheet: IHC dilution). The caption does not report the fixative (A10561-1 image caption). For cross-species work, mouse and rat reactivity is listed, but the pictured IHC tissue is human; for IF/ICC, no IF application, dilution, or image is listed (catalog applications/reactivity/IF data; A10561-1 image caption).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P42766 (RL35_HUMAN, Large ribosomal subunit protein uL29).
- Human Protein Atlas. RPL35 tissue IHC expression (reliability: Approved).
- Human Protein Atlas. RPL35 subcellular location (ICC-IF): Localized to the endoplasmic reticulum and cytosol..
- Human Protein Atlas. RPL35 antibody validation summary (1 antibodies).
- RPL35 downregulated by mechanical overloading promotes chondrocyte senescence and osteoarthritis development via Hedgehog-Gli1 signaling. Journal of orthopaedic translation 2024 — PMC11001632.
- GADD45GIP1 promotes osteosarcoma progression by modulating RPL35 ubiquitination and alleviating endoplasmic reticulum stress via the PERK/eIF2α pathway. Cancer cell international 2025 — PMC12220624.
- PubMed PMID:14702039 — UniProt-cited evidence.
- PubMed PMID:15164053 — UniProt-cited evidence.
- PubMed PMID:15489334 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for RPL35 (Large ribosomal subunit protein uL29). Expected localisation General cytoplasmic staining (HPA tissue IHC). Antigen retrieval: Citrate pH 6.0 HIER, 95–98 °C, 20 min (rule: cytoplasmic / membrane antigen). Validated IHC antibody: A10561-1 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Appendix (Glandular cells); negative Kidney. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded human lung carcinoma tissue; fixative not specified (datasheet A10561-1); 2. Fixation: Image fixative and duration unreported (datasheet A10561-1); 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-induced epitope retrieval in citrate buffer, pH 6.0 , 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-RPL35, 1:50-1:200 (datasheet A10561-1); 9. Primary incubation: Overnight at 4 °C (standard). Watch for: Fixation: Keep fixation consistent across sections. (standard IHC practice; not target-specific); Caveat: Staining and RNA show medium consistency (HPA tissue IHC); Isoform / epitope: No annotated isoforms; mature chain spans residues 2–123 (UniProt); Regulation: Low tissue specificity of RNA (HPA tissue RNA). Evidence: UniProt P42766, HPA tissue IHC · Approved, HPA ICC-IF · endoplasmic reticulum and cytosol., Datasheet A10561-1, PMC11001632. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/RPL35/immunohistochemistry-ihc-guide.