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

Design Immunohistochemistry for RICTOR

Plan paraffin section RICTOR IHC using the cytoplasmic tissue pattern and high hepatocyte staining as reference points (HPA tissue IHC). This guide covers fixation consistency (standard IHC practice), an antibody range of 0.5–1 µg/mL (datasheet A03195-1), and staining controls.

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 most tissues (HPA tissue IHC), antibody A03195-1, validated IHC image, and IHC protocol steps — Printable **RICTOR** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody A03195-1, controls and protocol steps. Open the full RICTOR IHC guide →

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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 most tissues (HPA tissue IHC)
	Staining pattern	Most tissues show cytoplasmic staining; hepatocytes stain high (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	Staining and RNA show only medium consistency (HPA tissue IHC)
	Regulation	RICTOR abundance regulation is unreported (UniProt)
	Isoform / epitope	3 isoforms; epitope coverage is unspecified (UniProt)

Section 1

Recommended RICTOR IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet A03195-1) is accompanied by 3 published RICTOR IHC protocols (PMC7912927; PMC11066283; PMC8442624).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A03195-1)
Fixation	Image fixative and duration unreported (datasheet A03195-1); 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 A03195-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A03195-1)
Primary antibody	Rabbit anti-RICTOR, 0.5-1μg/ml (datasheet A03195-1)
Primary incubation	Overnight at 4 °C (datasheet A03195-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A03195-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RICTOR-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 heat-mediated EDTA at pH 8.0 for the catalog antibody (datasheet A03195-1); the published protocols describe other retrieval conditions for their antibodies (PMC7912927; PMC11066283; PMC8442624).

Section 2

What Is the Expected RICTOR Staining Pattern?

RICTOR is associated with the cell membrane, endoplasmic reticulum membrane and lysosome membrane as part of mTORC2, despite having no annotated transmembrane segment (UniProt Q6R327: subcellular location, topology). In paraffin tissue sections, expect predominantly cytoplasmic staining in many cell types, with strong examples in hepatocytes, kidney tubule cells and esophageal squamous cells (HPA tissue IHC: ubiquitous cytoplasmic expression; High in each named cell type). HPA rates its tissue IHC profile Approved, with medium RNA–staining consistency and external verification pending (HPA tissue IHC: reliability).

What am I looking at on my slide?
Cytoplasmic staining in hepatocytes, kidney tubule cells or esophageal squamous cells, with interpretable cell boundaries (HPA tissue IHC: High in these cell types).	This fits the reported tissue pattern (HPA tissue IHC: ubiquitous cytoplasmic expression). Score the named cell population and its intensity, rather than treating every cell in the section as an equally informative control (general IHC interpretation practice).
Strong nuclear-only staining with little cytoplasmic signal in an otherwise readable section.	A nuclear-only pattern does not match HPA tissue IHC's cytoplasmic profile or UniProt's membrane-associated locations (HPA tissue IHC: cytoplasmic; UniProt Q6R327: subcellular location). Check antibody specificity and detection background before calling it RICTOR (general IHC practice).
Strong staining in a purported negative comparator, such as adipocytes or oral mucosal squamous cells (HPA tissue IHC: Not detected in those cell types).	Unexpected staining raises the possibility of cross-reactivity or endogenous detection activity (general IHC practice). Compare the exact cell type and tissue: esophageal squamous cells are High, while oral mucosal squamous cells are Not detected (HPA tissue IHC).
Diffuse chromogen across cells and extracellular areas, obscuring cytoplasmic boundaries.	This is difficult to score as the reported cytoplasmic pattern (HPA tissue IHC: cytoplasmic). Uneven washing, excessive detection signal or nonspecific binding can produce background (general IHC practice); assess a detection control before interpreting intensity.
No discernible staining in hepatocytes or kidney tubule cells despite intact morphology (HPA tissue IHC: High in both cell types).	The run may lack sensitivity or the sampled section may be uninformative (general IHC practice). HPA's High calls provide reference comparators, but its Approved rating has medium RNA–staining consistency and awaits external verification (HPA tissue IHC: reliability).

💡Expected RICTOR appearanceCall a positive result when cytoplasmic signal is readily visible in hepatocytes, kidney tubule cells or esophageal squamous cells, which HPA scores High; isolated nuclear staining or uniform extracellular deposit should prompt an artifact check (HPA tissue IHC: cytoplasmic, High in named cells; general IHC practice).

How each factor affects the staining
Cellular location	mTORC2 acts at plasma, endoplasmic reticulum and lysosome membranes, while tissue IHC is described broadly as cytoplasmic; routine chromogenic sections may show the broader pattern (UniProt Q6R327: subcellular location; HPA tissue IHC: cytoplasmic).
Tissue and cell selection	HPA reports High staining in liver hepatocytes and kidney tubule cells, but Not detected in adipocytes and ovarian stromal cells. Use the stated cell populations when comparing sections (HPA tissue IHC).
IHC evidence strength	The tissue profile is Approved with medium consistency between staining and RNA data, pending external verification; both listed RICTOR antibodies have IHC Approved status, without an Enhanced claim in this payload (HPA tissue IHC: reliability; HPA antibodies: IHC status).
IF/ICC Q: Should mitochondrial fluorescence define the IHC readout?	A: HPA calls mitochondria the approved ICC-IF location, whereas UniProt places mTORC2 at plasma, ER and lysosome membranes. Interpret this page's chromogenic tissue pattern from HPA tissue IHC; resolve the differing compartment calls in the separate IF/ICC guide (HPA subcellular ICC-IF; UniProt Q6R327; HPA tissue IHC).
Isoforms and epitope coverage	UniProt lists 3 isoforms, but the supplied evidence gives no catalog epitope or isoform-specific IHC result. Do not infer that a stain detects every isoform or that isoforms explain a negative section (UniProt Q6R327: isoforms).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No signal in a reference positive cell population.	The assay may have insufficient detectable signal, or the chosen cells may be absent from the section (general IHC practice).	Confirm hepatocytes or kidney tubule cells are present and assess a working positive control; HPA reports High staining in these populations (HPA tissue IHC). Review the catalog antibody's IHC-P conditions for the actual run (general IHC practice).
Only nuclear staining is apparent.	The pattern conflicts with HPA's cytoplasmic tissue profile and UniProt's membrane-associated annotation; nonspecific staining is possible (HPA tissue IHC; UniProt Q6R327; general IHC practice).	Inspect a detection control and compare the suspect section with a readable positive tissue. Reassess antibody specificity before assigning nuclear signal to RICTOR (general IHC practice).
Adipocytes or ovarian stromal cells stain strongly.	HPA reports RICTOR Not detected in those specific cell types; apparent positivity may reflect cross-reactivity or detection background (HPA tissue IHC; general IHC practice).	Verify cell identity and compare a detection control with an HPA High cell population on a comparable run (HPA tissue IHC: hepatocytes and kidney tubule cells High; general IHC practice).
Most of the section has diffuse brown deposit.	Background can result from nonspecific binding or excessive detection signal, making cellular intensity unreliable (general IHC practice).	Check background in a detection control, then review blocking, washing and detection conditions. Score RICTOR only where cell boundaries and cytoplasmic signal remain interpretable (general IHC practice; HPA tissue IHC: cytoplasmic).
A squamous tissue disagrees with the expected intensity.	Squamous cells differ by sampled site: esophagus is High, while oral mucosa and vagina are Not detected in HPA's tissue IHC data (HPA tissue IHC).	Confirm the tissue site and score the specified squamous cell population. Compare it with the matching HPA tissue observation before changing assay conditions (HPA tissue IHC; general IHC practice).
Fluorescent puncta appear mitochondrial, while the chromogenic section looks cytoplasmic.	HPA approves mitochondrial ICC-IF localisation and reports a broad cytoplasmic tissue IHC pattern; UniProt annotates mTORC2 at several membranes (HPA subcellular ICC-IF; HPA tissue IHC; UniProt Q6R327).	Record each assay and compartment separately. Use the tissue IHC pattern to judge this chromogenic result and consult the dedicated IF/ICC guide for fluorescence interpretation (HPA tissue IHC; HPA subcellular ICC-IF).

Source: UniProt Q6R327 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RICTOR staining

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.

A03195-1 · IHCIHC analysis of RICTOR using anti-RICTOR antibody (A03195-1). RICTOR was detected in paraffin-embedded section of human mammary cancer tissu

IF analysis of RICTOR using anti-RICTOR antibody (A03195-1). RICTOR was detected in immunocytochemical section of A431 cells. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/mL rabbit anti-RICTOR Antibody (A03195-1) overnight at 4°C. DyLight®488 conjugated Goat Anti-Rabbit IgG (BA1127) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A03195-1 · IFIF analysis of RICTOR using anti-RICTOR antibody (A03195-1). RICTOR was detected in immunocytochemical section of A431 cells. Enzyme antigen

M03195-1 · IHCMouse lung was stained with anti-Rictor rabbit antibody

M03195-1 · IHCHuman prostate was stained with anti-Rictor rabbit antibody

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000164327-RICTOR/tissue/cerebral+cortex#imid_9065428

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000164327-RICTOR/tissue/colon#imid_9065346

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000164327-RICTOR/tissue/liver#imid_9065339

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000164327-RICTOR/tissue/kidney#imid_9065356

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA037802; green = RICTOR, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

Real IHC / IF data behind the staining-pattern expectations in this section.

Sample controls for RICTOR IHC & IF

🧪Run esophagus first and expect staining in its squamous epithelial cells (HPA: High); use oral mucosa squamous epithelial cells as the negative tissue (HPA: Not detected). On the positive slide, cells without DAB staining provide an internal background reference, but HPA does not establish which esophageal cell types are truly 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 no-primary (secondary-only) and nonimmune rabbit IgG isotype controls (selected-SKU caption: rabbit primary), plus RICTOR-knockout material as a biological negative. With the caption’s biotin-based SABC/DAB detection, control for endogenous peroxidase and biotin (selected-SKU caption: SABC/DAB).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the fixative for the selected-SKU paraffin-section caption is unreported (selected-SKU caption: A03195-1). Heat retrieval in EDTA at pH 8.0 is a documented starting condition, but retrieval dependency has not been established (selected-SKU caption: EDTA pH 8.0). The supplied evidence does not establish whether frozen sections or IF are easier than paraffin IHC, or identify an esophagus-specific artefact; ICC-IF images are available for RICTOR (HPA: subcellular images).

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

Tissue	Cell type	Level	Evidence	Source
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

Tissue	Cell type	Level	Evidence	Source
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

Troubleshooting RICTOR staining in paraffin sections requires attention to retrieval, compartment patterns, controls and cell level scoring.

How should I optimize retrieval when RICTOR staining is weak in paraffin sections?

Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet A03195-1). Keep heating and cooling conditions consistent across sections, then compare signal with a matched section processed without primary antibody to identify detection background (standard IHC practice). The product image used 1 μg/mL primary antibody overnight at 4°C, so preserve those conditions while evaluating retrieval (datasheet A03195-1). If staining remains weak, test a different retrieval condition on adjacent sections as a documented fallback, checking tissue morphology and background before adopting it (standard IHC practice).

Could fixation explain variable RICTOR staining between paraffin blocks?

RICTOR specific sensitivity to fixation is unknown because the selected tissue image states that the section was paraffin embedded but does not report its fixative (datasheet A03195-1). Record the fixative, fixation duration and processing history for each block before comparing staining across specimens (standard IHC practice). Compare sections processed together using EDTA retrieval at pH 8.0 and the same 1 μg/mL primary concentration to reduce technical variation (datasheet A03195-1; standard IHC practice). If differences persist, repeat staining on independently processed sections and review morphology alongside antibody signal; do not assign the difference to a RICTOR specific fixation effect without direct evidence (standard IHC practice).

How should I interpret punctate or diffuse RICTOR staining in tissue?

Assess staining within identified cells before assigning it to a compartment: tissue IHC shows widespread cytoplasmic expression, while the subcellular IF record reports mitochondrial localization (HPA tissue IHC; HPA subcellular). RICTOR has no annotated transmembrane segment, although mTORC2 localizes at the plasma membrane, endoplasmic reticulum membrane and lysosomes (UniProt Q6R327 topology and subcellular record). Thus, a punctate DAB pattern alone cannot establish a particular organelle; compare serial sections and cell morphology, and use a compartment marker in a separate validation experiment (standard IHC practice). Treat strong nuclear only staining cautiously because neither supplied localization record identifies the nucleus as its principal location (HPA tissue IHC; HPA subcellular).

Could epitope choice explain inconsistent RICTOR staining?

RICTOR has 3 listed isoforms, but the supplied antibody caption does not identify the immunogen or establish which isoforms its epitope recognizes (UniProt Q6R327 isoforms; datasheet A03195-1). Its record also lists multiple modified residues, including phosphoserines at positions 21, 35 and 265, without linking those sites to this antibody's binding (UniProt Q6R327 modified residues; datasheet A03195-1). Review the antibody's documented immunogen or epitope before interpreting absent tissue signal as absence of every isoform (standard IHC practice). If that information remains unavailable, compare staining with an independently validated reagent and maintain identical retrieval and detection conditions across sections (standard IHC practice).

How can I check RICTOR localization by IF alongside tissue IHC?

Pair the RICTOR channel with a marker that identifies the expected cell population, such as a validated epithelial marker when examining glandular cells, and confirm marker specificity separately (HPA tissue IHC: high in several glandular cell populations; standard IF practice). Select fluorophores in a spectral region with low tissue autofluorescence, and inspect unstained and single stained controls before judging apparent overlap (standard IF practice). Because RICTOR lacks an annotated transmembrane segment and mTORC2 associates with membranes, optimize permeabilisation to reach the antibody's actual epitope side without assuming a membrane spanning topology (UniProt Q6R327 topology and subcellular record; standard IF practice). Interpret IF patterns alongside cell identity and compartment markers rather than equating fluorescence overlap with direct molecular interaction (standard IF practice).

What should I change when RICTOR DAB staining is diffuse or nonspecific?

First compare a no primary control with the stained section to separate antibody dependent signal from endogenous enzyme activity and detection background (standard IHC practice). The selected product image used 10% goat serum block, biotinylated goat anti rabbit secondary for 30 minutes at 37°C, streptavidin biotin detection and DAB (datasheet A03195-1). With this detection scheme, check endogenous biotin and peroxidase controls, then review blocking, washing and development time on matched sections (standard IHC practice). Reduce primary concentration only after confirming that background follows the primary antibody, and retain a known positive tissue section in each comparison (standard IHC practice; HPA tissue IHC: high in kidney tubule cells).

How should I score RICTOR IHC across heterogeneous specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring; HPA reports widespread cytoplasmic staining but variable intensity among cell types (HPA tissue IHC). For a defined population, record the percentage of positive cells and staining intensity, then calculate an H score as 1 × weak + 2 × moderate + 3 × strong percentage, with a possible range of 0–300 (standard IHC practice). Normalize comparisons to the number of evaluable cells or tissue area, and report compartment specific scores separately rather than pooling unlike populations (standard IHC practice). Apply the same threshold, retrieval, staining batch and image settings to all sections, excluding folds, edges and nonviable regions by prespecified rules (standard IHC practice).

How can I distinguish true RICTOR staining from tissue artefact?

Favor signal within intact cells and a plausible cytoplasmic distribution, using kidney tubule cells as one documented high staining reference and adipocytes as a documented undetected population (HPA tissue IHC). A sharply stained section edge, necrotic area or tissue fold should prompt review of morphology and adjacent well preserved areas before assigning a biological pattern (standard IHC practice). Test suspicious DAB deposition with no primary and endogenous peroxidase controls, especially when staining does not follow cell boundaries (standard IHC practice). RICTOR is an mTORC2 component, but its IHC abundance alone cannot establish kinase activity; assess pathway readouts separately when that is the research question (UniProt Q6R327 function; standard IHC interpretation).

Boster reagents

Best RICTOR / Rapamycin-insensitive companion of mTOR IHC Antibodies

IHC images document RICTOR staining in human mammary cancer and prostate tissue and mouse lung; an IF image documents staining in A431 cells (catalog image captions).

Real IHC data

Anti-RICTOR Antibody ®

Cat # A03195-1

View product →

Real IHC data Mouse lung was stained with anti-Rictor rabbit antibody

Anti-Rictor Rabbit Monoclonal Antibody

Cat # M03195-1

View product →

A03195-1 has IHC data from paraffin-embedded human mammary cancer tissue and IF data from A431 cells (catalog image captions). M03195-1 has IHC images from mouse lung and human prostate and lists IF among its applications (catalog image captions and application list).

Which to pick: For human paraffin-section IHC, choose A03195-1: its IHC image documents EDTA retrieval in a paraffin-embedded section, with fixative unreported (A03195-1 IHC caption). For IF/ICC, choose A03195-1 because its application list includes both and its IF image shows A431 cells (A03195-1 application list and IF caption). For human and mouse tissue IHC, choose the monoclonal M03195-1; its images show mouse lung and human prostate, but their processing and fixative are unreported (M03195-1 description, reactivity and IHC captions).

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

References

UniProt Consortium. UniProt entry Q6R327 (RICTR_HUMAN, Rapamycin-insensitive companion of mTOR).
Human Protein Atlas. RICTOR tissue IHC expression (reliability: Approved).
Human Protein Atlas. RICTOR subcellular location (ICC-IF): Localized to the mitochondria..
Human Protein Atlas. RICTOR antibody validation summary (2 antibodies).
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.
An Immunohistochemical Study of the PTEN/AKT Pathway Involvement in Canine and Feline Mammary Tumors. Animals : an open access journal from MDPI 2021 — PMC7912927.
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.
Rictor Activates Cav 1 Through the Akt Signaling Pathway to Inhibit the Apoptosis of Gastric Cancer Cells. Frontiers in oncology 2021 — PMC8442624.
PubMed PMID:15268862 — UniProt-cited evidence.
PubMed PMID:17974005 — UniProt-cited evidence.
PubMed PMID:15372022 — UniProt-cited evidence.