RICTOR / Rapamycin-insensitive companion of mTOR · IHC design guide

Design Immunohistochemistry for RICTOR

Plan RICTOR chromogenic IHC in paraffin sections using the reported cytoplasmic tissue pattern (HPA tissue IHC). This guide covers fixation, staining controls and interpretation of tissue differences.

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
Immunohistochemistry protocol sheet for RICTOR (IHC for RICTOR): expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC), antibody A03195-1, validated IHC image, and IHC protocol steps
Printable RICTOR IHC protocol sheet — expected localisation Cytoplasmic staining in tissue sections (HPA tissue IHC), antibody A03195-1, controls and protocol steps. Open the full RICTOR IHC guide →

RICTOR 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 tissue sections (HPA tissue IHC)
Staining pattern Cytoplasmic in most tissues; high in some glandular cells (HPA tissue IHC)
Antigen retrieval EDTA pH 8.0 HIER, heat-mediated (datasheet A03195-1)
Positive control ⓘ Adrenal gland+4 more · see all
Negative control ⓘ Adipose tissue+4 more · see all
Important caveats
Reasons your staining may differ from the expected pattern.
Fixation Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A03195-1)
Caveat Squamous cells stain high in esophagus but are undetected in oral mucosa (HPA tissue IHC)
Regulation GSK3/FBXW7 promotes RICTOR degradation (UniProt)
Isoform / epitope Three isoforms; check antibody epitope coverage (UniProt)
Section 1

Recommended RICTOR IHC & IF Protocols

The catalog antibody’s IHC-P protocol uses heat-mediated EDTA retrieval at pH 8.0 (datasheet). Three published RICTOR IHC protocols provide tissue-specific comparisons (PMC7912927; PMC11066283; PMC8442624).

Recommended immunohistochemistry (IHC-P) protocol parameters
SampleParaffin-embedded human mammary cancer tissue; fixative not specified (datasheet A03195-1)
FixationImage fixative and duration unreported (datasheet A03195-1); verify before use.
Sectioning4–5 µm sections on charged slides (standard)
DeparaffinisationXylene, graded ethanol series to water (standard)
Antigen retrievalHeat retrieval: EDTA pH 8.0 (datasheet A03195-1); 20 min, 95–100 °C (standard)
Peroxidase block3% H2O2, 10 min, room temperature (standard)
Blocking10% goat serum (datasheet A03195-1)
Primary antibodyRabbit anti-RICTOR, 0.5-1μg/ml (datasheet A03195-1)
Primary incubationOvernight at 4 °C (datasheet A03195-1)
DetectionStreptavidin-biotin complex (SABC), DAB chromogen (datasheet A03195-1)
CounterstainHematoxylin, blue, dehydrate and mount (standard)
Expected resultRICTOR-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous cytoplasmic expression in most tissues. No signal in the no-primary control.
💡Decision noteStart with EDTA pH 8.0 for the catalog antibody (datasheet); the published citrate pH 6.0 conditions used different antibodies (PMC7912927; PMC11066283).
Section 2

What Is the Expected RICTOR Staining Pattern?

RICTOR staining in paraffin sections should be predominantly cytoplasmic in many cell types (HPA tissue IHC: ubiquitous cytoplasmic expression). High signal is reported in hepatocytes, kidney tubule cells, cardiomyocytes and several glandular epithelia (HPA tissue IHC: High). RICTOR has no transmembrane segment, although mTORC2 associates with the cell, endoplasmic reticulum and lysosome membranes (UniProt Q6R327 topology and subcellular location). The tissue IHC profile is Approved, with medium staining–RNA consistency and external verification pending (HPA tissue IHC).

What am I looking at on my slide?
Cytoplasmic signal is strong in hepatocytes or kidney tubule cells, with cell outlines and nuclei still readable.This fits the reported compartment and two High cell populations (HPA tissue IHC: cytoplasmic profile; High in hepatocytes and kidney tubule cells). Membrane-associated mTORC2 does not require a sharp membrane rim in chromogenic sections (UniProt Q6R327 subcellular location; general IHC interpretation). Compare stained cells with nearby tissue architecture before scoring intensity.
The dominant signal is confined to nuclei, extracellular material or a continuous tissue-edge deposit.Those patterns depart from the reported cytoplasmic tissue pattern (HPA tissue IHC). Check whether the signal follows tissue boundaries, damaged areas or counterstain rather than intact cells (general IHC practice). A discordant compartment is a reason to investigate an artefact; location alone cannot identify its cause.
Adipocytes, ovarian stromal cells or soft-tissue fibroblasts stain as strongly as the positive comparison cells.These populations are listed as Not detected in the supplied tissue observations (HPA tissue IHC). Recheck cell identification and compare controls; cross-reactivity or endogenous detection activity is possible (general IHC practice). An unexpected positive is not conclusive by itself because HPA rates the tissue profile Approved with medium consistency and pending external verification (HPA tissue IHC).
Color is diffuse across cells, stroma and empty areas, obscuring boundaries.A uniform haze cannot be scored as the reported cytoplasmic cell staining (HPA tissue IHC; general IHC interpretation). Inspect a no-primary control and the distribution of the precipitate (general IHC practice). Background that persists without primary antibody points toward the detection workflow rather than specific RICTOR binding (general IHC practice).
A hepatocyte or kidney tubule positive-control section has no detectable signal.Both cell populations are reported High (HPA tissue IHC), so an absent result calls the run into question. Verify that the expected cells are present, then review retrieval, primary incubation and chromogenic detection using the validated IHC workflow (general IHC practice). A single negative section does not establish biological absence.
💡Expected RICTOR appearanceCall a result positive when intact cells show predominantly cytoplasmic staining, potentially strong in hepatocytes or kidney tubules (HPA tissue IHC: cytoplasmic; High); isolated nuclear color, extracellular deposits or uniform haze are suspect (general IHC interpretation).
How each factor affects the staining
Tissue and cell selectionUse an HPA High cell population, such as hepatocytes or kidney tubule cells, as a positive comparison (HPA tissue IHC). Adipocytes and ovarian stromal cells are reported Not detected (HPA tissue IHC). These are observed patterns, not guaranteed outcomes for every section or antibody (HPA tissue IHC: Approved; medium consistency, pending external verification).
Compartment and topologyRICTOR has no annotated transmembrane segment, while mTORC2 is described at plasma, endoplasmic reticulum and lysosome membranes (UniProt Q6R327). Interpret the supplied tissue IHC reference as cytoplasmic staining (HPA tissue IHC); do not require a crisp plasma-membrane outline as the criterion for positivity.
Antibody validation and isoformsHPA037802 and HPA037803 have Approved IHC status; the supplied record does not label either IHC result Enhanced (HPA antibodies). UniProt lists three RICTOR isoforms (UniProt Q6R327). The payload gives no epitope coverage for either antibody, so it cannot establish whether staining measures every isoform.
IF/ICC Q&A: should mitochondrial puncta appear in IHC?HPA reports mitochondria as the approved main location in ICC-IF, while its tissue IHC summary reports ubiquitous cytoplasmic expression (HPA subcellular ICC-IF; HPA tissue IHC). Treat the ICC-IF observation as application-specific context, not a required chromogenic IHC pattern. This section supplies no IF/ICC protocol option.
Why is my staining missing, weak or wrong?
SituationLikely causeNext action
Expected positive cells are blank while the section has intact morphology.The run may have inadequate primary staining or detection; absence is difficult to interpret without a positive comparison (general IHC practice; HPA tissue IHC: High in hepatocytes and kidney tubule cells).Run a reported High tissue alongside the sample, and check the IHC-validated antibody's documented retrieval, incubation and detection conditions (general IHC practice). Record the observed cell types before concluding that RICTOR is absent.
A no-primary control develops chromogen.Primary-independent detection activity or background is possible (general IHC practice). The HPA tissue profile cannot identify that workflow cause (HPA tissue IHC).Review the detection-system blocking and wash steps and repeat the no-primary control (general IHC practice). Score sample staining only after primary-independent color is controlled.
Staining is widespread but cell boundaries are hard to distinguish.Diffuse background may obscure the cytoplasmic pattern reported for tissue sections (HPA tissue IHC; general IHC practice).Compare with a no-primary section, review washing and primary-antibody concentration within the validated IHC workflow, and retain settings that preserve cellular detail (general IHC practice). Do not score haze as cell-specific staining.
Only nuclei or tissue edges show strong color.This distribution conflicts with the reported cytoplasmic tissue pattern (HPA tissue IHC); localisation alone does not prove which artefact occurred.Inspect tissue integrity and control sections, then repeat with the IHC-validated antibody under its documented conditions (general IHC practice). Require reproducible staining in identifiable cells before assigning a RICTOR pattern.
A reported Not detected cell population stains prominently.Cell misidentification, cross-reactivity or endogenous detection activity are possibilities (general IHC practice; HPA tissue IHC: Not detected in adipocytes, ovarian stromal cells and soft-tissue fibroblasts).Verify cell identity on the counterstained section and compare a no-primary control with a reported High population in the same run (general IHC practice; HPA tissue IHC). Treat a persistent difference from HPA as unresolved rather than automatically dismissing it.
Mitochondrial-looking puncta appear, but the chromogenic tissue pattern is unclear.HPA assigns mitochondria as an approved ICC-IF location, whereas its tissue IHC summary describes cytoplasmic staining (HPA subcellular ICC-IF; HPA tissue IHC). The two observations do not establish a punctate IHC criterion.Judge the IHC slide against identifiable cytoplasmic staining in reported positive cells and its controls (HPA tissue IHC; general IHC practice). Record puncta separately instead of treating them as proof of mitochondrial localisation in the paraffin section.

Sample controls for RICTOR IHC & IF

🧪Run esophagus first; its squamous epithelial cells should stain (HPA: High in esophageal squamous epithelial cells). Use oral mucosa as the negative tissue (HPA: Not detected in oral mucosal squamous epithelial cells); on the esophagus slide, treat adjacent non-epithelial cells as background comparators rather than assuming they are RICTOR-negative.
Positive control tissue: Adrenal gland (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RICTOR in A-431, U-251MG, U2OS, hTERT-RPE1 (serum starved), with annotated localisation: Mitochondria (approved) (HPA subcellular).
Technical controls: Include a no-primary, secondary-only section, a species- and class-matched rabbit IgG isotype control (caption: rabbit primary antibody), and RICTOR-knockout tissue or a validated immunogen-peptide block as a biological specificity control. For chromogenic detection, quench endogenous peroxidase and assess endogenous biotin background because the caption uses biotinylated secondary antibody, streptavidin–biotin complex, and DAB (selected-SKU caption: A03195-1).
⚠️Feasibility: A target-specific fixation window and fixation effect are unreported, and the fixative in the selected-SKU paraffin-section caption is unreported (selected-SKU caption: A03195-1). The demonstrated IHC procedure uses heat-mediated EDTA retrieval at pH 8.0, but the evidence does not establish whether retrieval is required or whether frozen sections or IF are easier (selected-SKU caption: A03195-1). In esophagus, check surface and section-edge DAB deposits against the no-primary control before scoring squamous epithelial staining (HPA: High in esophageal squamous epithelial cells).

HPA tissue IHC evidence for RICTOR

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium consistency between antibody staining and RNA expression data. Pending external verification.). Rows are taken directly from the HPA tissue chart — click any row's HPA link to view the source.

Positive expression · recommended positive controls

TissueCell typeLevelEvidenceSource
Adrenal gland Glandular cells High Protein (IHC) HPA →
Duodenum Glandular cells High Protein (IHC) HPA →
Epididymis Glandular cells High Protein (IHC) HPA →
Esophagus Squamous epithelial cells High Protein (IHC) HPA →
Gallbladder Glandular cells High Protein (IHC) HPA →

Undetected expression · recommended negative controls

TissueCell typeLevelEvidenceSource
Adipose tissue Adipocytes Not detected Protein (IHC) HPA →
Oral mucosa Squamous epithelial cells Not detected Protein (IHC) HPA →
Ovary Ovarian stroma cells Not detected Protein (IHC) HPA →
Soft tissue Fibroblasts Not detected Protein (IHC) HPA →
Vagina Squamous epithelial cells Not detected Protein (IHC) HPA →
Section 3

Advanced RICTOR IHC Tips

Troubleshoot RICTOR staining in paraffin sections by checking retrieval, compartment, cell identity and controls before comparing chromogenic signal across samples.

What should I change when RICTOR staining is weak after antigen retrieval?
Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet A03195-1). The demonstrated paraffin-section workflow then used 10% goat serum, 1 µg/mL primary antibody overnight at 4°C, and DAB detection (datasheet A03195-1). If signal remains weak, compare a modestly longer heating interval with the original interval on adjacent sections, while holding antibody concentration and detection constant (standard IHC practice). Check that the comparison preserves tissue morphology, and include a no-primary control so extra heating is not mistaken for improved specific staining (standard IHC practice).
Could fixation explain inconsistent RICTOR staining between paraffin blocks?
RICTOR-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state a fixative (datasheet A03195-1). Record each block’s fixative and fixation duration, then compare similarly processed sections using the same pH 8.0 EDTA retrieval and antibody conditions (datasheet A03195-1; standard IHC practice). If a block stains weakly, inspect morphology and a concurrently stained control before changing retrieval, because inconsistent processing can affect antigen access and chromogenic signal (standard IHC practice). Do not infer a preferred fixative from RICTOR’s membrane-associated locations or phosphorylation sites (UniProt Q6R327); neither establishes its fixation response.
Should RICTOR appear at membranes, in cytoplasm, or near mitochondria?
Assess the pattern within cells rather than requiring a sharp membrane outline: RICTOR is an mTORC2 component associated with plasma, endoplasmic reticulum and lysosome membranes, yet has no annotated transmembrane segment (UniProt Q6R327). Tissue IHC is reported as broadly cytoplasmic (HPA tissue IHC), while approved ICC/IF localisation is mitochondrial (HPA subcellular). These observations support recording diffuse, punctate and peripheral cytoplasmic staining separately; chromogenic IHC alone cannot resolve which organelle supplies a punctum (standard IHC practice). If a result is exclusively nuclear, compare staining with a no-primary control and an independently validated antibody before assigning it to RICTOR (standard IHC practice; UniProt Q6R327).
Can isoforms or epitope changes account for discrepant RICTOR IHC?
RICTOR has 3 annotated isoforms and multiple modified residues, including phosphoserines and acetyllysines (UniProt Q6R327). The supplied IHC caption does not identify this antibody’s epitope or establish whether it recognises all isoforms (datasheet A03195-1). Map the immunogen against the annotated isoform sequences if epitope information becomes available, and avoid interpreting a staining difference as isoform switching without independent evidence (standard IHC practice; UniProt Q6R327). When comparing sections, keep pH 8.0 EDTA retrieval and 1 µg/mL primary antibody constant so processing changes do not confound the comparison (datasheet A03195-1).
How should I investigate an unexpected RICTOR pattern by multiplex IF?
Use this as a separate ICC/IF validation experiment: HPA reports approved mitochondrial localisation in imaged cell lines, whereas UniProt describes mTORC2 activity at plasma, endoplasmic reticulum and lysosome membranes (HPA subcellular; UniProt Q6R327). Pair RICTOR with a marker identifying the expected cell type and, if localisation is the question, an appropriate organelle marker (standard IF practice). Choose spectrally separated fluorophores and place the weaker signal in a channel with lower tissue autofluorescence, checking single-stain controls (standard IF practice). Because RICTOR has no annotated transmembrane segment, optimise permeabilisation to expose its intracellular epitope, whose position is unspecified here (UniProt Q6R327; standard IF practice).
How can I reduce diffuse or granular DAB background?
Run no-primary and secondary-only controls, and inspect whether pigment, tissue edges or damaged regions retain color independently of primary antibody (standard IHC practice). The demonstrated workflow used 10% goat serum before 1 µg/mL rabbit primary antibody overnight at 4°C, followed by a biotinylated secondary, avidin-biotin detection and DAB (datasheet A03195-1). For diffuse background, compare shorter primary incubation or lower concentration on matched sections, changing one variable at a time (standard IHC practice). For granular background with avidin-biotin detection, check endogenous biotin and apply appropriate blocking; include a peroxidase block before DAB as a general chromogenic IHC step (standard IHC practice).
How should I score RICTOR staining across tissue samples? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and region before scoring, excluding folds, necrosis and section edges (standard IHC practice). For cytoplasmic RICTOR, report the percentage of positive target cells and an H-score from 0–300 based on staining intensity and the fraction at each intensity; keep thresholds fixed across slides (HPA tissue IHC; standard IHC practice). Normalise positive-cell counts to the number of evaluable target cells, or signal area to evaluable tissue area, rather than comparing raw brown area across differently sized sections (standard IHC practice). Record block processing and staining batch, and score matched controls alongside specimens because the HPA tissue profile has medium RNA-to-staining consistency and awaits external verification (HPA tissue IHC; standard IHC practice).
When is a strong RICTOR signal likely to be artefactual?
Check cell identity and compartment first: HPA reports high staining in kidney tubular cells and no detected staining in adipocytes, while its overall tissue pattern is cytoplasmic (HPA tissue IHC). An exclusively nuclear signal warrants scrutiny against RICTOR’s annotated membrane-associated mTORC2 localisation and the separate mitochondrial ICC/IF report (UniProt Q6R327; HPA subcellular). Treat signal concentrated at section edges or necrotic areas, or present in a no-primary control, as suspect until matched controls resolve it (standard IHC practice). If DAB appears without primary antibody, investigate endogenous peroxidase or detection-reagent background before calling cells RICTOR-positive (standard IHC practice).
Boster reagents

Best RICTOR / Rapamycin-insensitive companion of mTOR IHC Antibodies

Two anti-RICTOR antibodies have tissue IHC images: human mammary cancer sections for A03195-1, and mouse lung and human prostate for M03195-1 (catalog IHC captions). A03195-1 also has A431 cell IF data (catalog IF caption).

Real IHC data IHC analysis of RICTOR using anti-RICTOR antibody (A03195-1). RICTOR was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-RICTOR Antibody (A03195-1) overnight at 4°C. Biotinylated goat anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using Strepavidin-Biotin-Complex (SABC) (Catalog # SA1022) with DAB as the chromogen.
Anti-RICTOR Antibody ®
Cat # A03195-1
Real IHC data Mouse lung was stained with anti-Rictor rabbit antibody
Anti-Rictor Rabbit Monoclonal Antibody
Cat # M03195-1

A03195-1 will render with IHC staining of a paraffin-embedded human mammary cancer section; its separate IF image shows A431 cells (A03195-1 image captions). M03195-1 will render with mouse lung IHC staining; another catalog caption reports human prostate staining (M03195-1 IHC captions).

Which to pick: Choose A03195-1 for human paraffin-section IHC because its own image documents that preparation; the fixative is unreported (A03195-1 IHC caption). For IF/ICC, choose A03195-1 when working with human samples: IF and ICC are listed applications, and its IF image shows A431 cells (A03195-1 applications and IF caption). For human or mouse tissue IHC, consider M03195-1: it is monoclonal, lists both species as reactive, and has mouse lung and human prostate staining captions; their processing and fixatives are unreported (M03195-1 catalog description, reactivity and IHC captions).

Each figure is that product's own IHC / IF validation image from its datasheet.

References

  1. UniProt Consortium. UniProt entry Q6R327 (RICTR_HUMAN, Rapamycin-insensitive companion of mTOR).
  2. Human Protein Atlas. RICTOR tissue IHC expression (reliability: Approved).
  3. Human Protein Atlas. RICTOR subcellular location (ICC-IF): Localized to the mitochondria..
  4. Human Protein Atlas. RICTOR antibody validation summary (2 antibodies).
  5. YTHDF3 Mediates the Occurrence and Development of Breast Cancer by Regulating Glycolysis Through the mTOR-HIF1α-LHDA Axis. Journal of cellular and molecular medicine 2026 — PMC13111412.
  6. An Immunohistochemical Study of the PTEN/AKT Pathway Involvement in Canine and Feline Mammary Tumors. Animals : an open access journal from MDPI 2021 — PMC7912927.
  7. RICTOR amplification is associated with Rictor membrane staining and does not correlate with PD-L1 expression in lung squamous cell carcinoma. Pathology oncology research : POR 2024 — PMC11066283.
  8. Rictor Activates Cav 1 Through the Akt Signaling Pathway to Inhibit the Apoptosis of Gastric Cancer Cells. Frontiers in oncology 2021 — PMC8442624.
  9. PubMed PMID:15268862 — UniProt-cited evidence.
  10. PubMed PMID:17974005 — UniProt-cited evidence.
  11. PubMed PMID:15372022 — UniProt-cited evidence.