RASAL1 / RasGAP-activating-like protein 1 · IHC design guide

Design Immunohistochemistry for RASAL1

Use adrenal or parathyroid glandular cells as high-staining references for RASAL1 paraffin IHC (HPA tissue IHC). Start the catalog antibody at 0.5–1 μg/ml (datasheet A06423-2), then assess membranous and cytoplasmic staining in the intended cell population (HPA tissue IHC).

Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use

Immunohistochemistry protocol sheet for RASAL1 (IHC for RASAL1): expected localisation Membranous and cytoplasmic (HPA tissue IHC), antibody A06423-2, validated IHC image, and IHC protocol steps — Printable **RASAL1** IHC protocol sheet — expected localisation Membranous and cytoplasmic (HPA tissue IHC), antibody A06423-2, controls and protocol steps. Open the full RASAL1 IHC guide →

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RASAL1 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	Membranous and cytoplasmic (HPA tissue IHC)
	Staining pattern	Membranous and cytoplasmic staining in glandular cells (HPA tissue IHC)
	Antigen retrieval	Citrate pH 6 HIER, heat-mediated (datasheet A06423-2)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	Liver+3 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	Skin Langerhans cells may confound epithelial scoring (HPA tissue IHC)
	Regulation	High in adrenal and parathyroid glands (HPA tissue IHC)
	Isoform / epitope	4 isoforms; confirm epitope coverage across variants (UniProt)

Section 1

Recommended RASAL1 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet A06423-2) and two published chromogenic IHC protocols (PMC10192437; PMC11312928) provide starting points for RASAL1 staining.

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded rat intestine tissue; fixative not specified (datasheet A06423-2)
Fixation	Image fixative and duration unreported (datasheet A06423-2); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Citrate pH 6, 20 min (datasheet A06423-2)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A06423-2)
Primary antibody	Rabbit anti-RASAL1, 0.5-1μg/ml (datasheet A06423-2)
Primary incubation	Overnight at 4 °C (datasheet A06423-2)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A06423-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	RASAL1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Membranous and cytoplasmic expression in several tissues, highly expressed in parathyroid and adrenal gland. No signal in the no-primary control.

💡Decision noteStart with heat-mediated citrate retrieval at pH 6 (datasheet A06423-2); a published tissue microarray protocol specifies 10 mM citrate for 15 min (PMC10192437).

Section 2

What Is the Expected RASAL1 Staining Pattern?

RASAL1 staining is expected at cell membranes and in cytoplasm across several tissues, with strong HPA staining in selected glandular and squamous epithelial cells (HPA: tissue IHC profile). Plasma membrane and cytosol localization is supported by ICC-IF images (HPA: approved subcellular locations). RASAL1 has no transmembrane segment (UniProt O95294 topology). The HPA tissue profile has Enhanced reliability, medium staining–RNA consistency, and pending external verification (HPA: tissue IHC reliability).

What am I looking at on my slide?
Membranous and cytoplasmic staining in adrenal or parathyroid glandular cells.	This matches high staining in those cell populations (HPA: tissue IHC). Interpret intensity in the identified cells, since an organ-wide score could obscure which cells are positive (general IHC practice).
Predominantly nuclear staining, with little membrane or cytoplasmic signal.	A nuclear-only pattern conflicts with the reported tissue and ICC-IF locations (HPA: tissue IHC; HPA: approved plasma membrane and cytosol). Review specificity and detection controls before assigning it to RASAL1 (general IHC practice).
Strong staining in cells expected to be unstained.	For example, cholangiocytes and lymph-node germinal center cells were not detected in the HPA tissue profile (HPA: tissue IHC). Cross-reactivity or endogenous detection activity is possible; these findings alone do not establish its cause (general IHC practice).
Diffuse chromogen across cells and surrounding tissue.	A pattern lacking discernible cell boundaries is difficult to score as the reported membranous and cytoplasmic distribution (HPA: tissue IHC). Check background and detection controls before interpreting weak cellular staining (general IHC practice).
No staining in glandular cells of a known-positive control section.	Adrenal and parathyroid glandular cells show high staining in the HPA profile (HPA: tissue IHC). A negative result there warrants a check of section quality and the IHC workflow before a test section is called negative (general IHC practice).

💡Expected RASAL1 appearanceCall a section positive when identifiable cells show membranous and cytoplasmic staining, especially strong staining in the listed glandular or squamous epithelial populations (HPA: tissue IHC); diffuse background or isolated nuclear staining is an atypical result requiring controls (HPA: tissue IHC; general IHC practice).

How each factor affects the staining
Which cells offer positive and negative comparisons?	High staining is reported in adrenal, parathyroid, salivary and stomach glandular cells and in esophageal, oral and vaginal squamous cells (HPA: tissue IHC). HPA reports no detection in liver cholangiocytes, lymph-node germinal center cells, ovarian stroma cells or spleen red-pulp cells; compare the specified cells, rather than whole organs (HPA: tissue IHC).
What does the localization evidence support?	Tissue IHC reports membranous and cytoplasmic expression (HPA: tissue IHC). ICC-IF places RASAL1 mainly at the plasma membrane and in cytosol, with additional cell-junction localization (HPA: approved subcellular locations). The locations are compatible with a protein lacking a transmembrane segment; that topology does not make every membrane signal specific (UniProt O95294 topology; general IHC practice).
Do isoforms or processing establish a staining pattern?	UniProt lists 4 isoforms and one annotated chain spanning residues 1–804, with no signal peptide or propeptide (UniProt O95294 isoforms and processing). These entries do not identify the antibody epitope or establish isoform-specific staining (UniProt O95294 isoforms; HPA: antibody validation summary).
How strong is the tissue evidence?	The HPA tissue profile is Enhanced but has medium consistency with RNA expression and awaits external verification (HPA: tissue IHC reliability). The listed antibody, HPA041650, has Enhanced IHC validation (HPA: antibody validation summary). Use the reported pattern as a reference and assess unexpected staining with controls (general IHC practice).
What should IF/ICC show?	Expect mainly plasma membrane and cytosol signal, with possible cell-junction signal, as reported in ICC-IF (HPA: approved subcellular locations). IF/ICC has a separate guide; this IHC section supplies no IF/ICC protocol (guide scope).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Positive-control glandular cells show no chromogen.	A failed staining run or unsuitable control section is possible; those cells stain strongly in the HPA profile (HPA: tissue IHC; general IHC practice).	Check tissue preservation, antigen retrieval, primary-antibody application and detection reagents against the catalog IHC-P procedure before interpreting study sections (general IHC practice). No RASAL1-specific retrieval sensitivity is established by these sources.
The whole section has diffuse background.	Nonspecific binding or detection-system background is possible (general IHC practice). Diffuse signal does not match the described cellular pattern (HPA: tissue IHC).	Inspect the negative reagent control and review blocking, washes and detection steps under the validated IHC workflow (general IHC practice). Score only distinguishable cellular staining.
Staining appears mainly in nuclei.	The compartment conflicts with reported membranous and cytoplasmic IHC and approved ICC-IF locations (HPA: tissue IHC; HPA: subcellular).	Confirm compartment assignment using the counterstain and cell boundaries, then compare positive and negative controls and review antibody specificity (general IHC practice).
Cholangiocytes or germinal center cells stain strongly.	Those specific populations were not detected in the HPA profile; cross-reactivity or endogenous detection activity is possible (HPA: tissue IHC; general IHC practice).	Verify cell identity and compare a negative reagent control; assess any detection-system background before treating the signal as RASAL1 (general IHC practice).
Only weak staining appears in an expected high-staining population.	The section or staining workflow may underperform; HPA reports high staining in the specified cells, but its profile has medium RNA consistency (HPA: tissue IHC).	Compare a concurrently stained positive control and review the catalog IHC-P procedure, including retrieval and dilution (general IHC practice). Do not infer a RASAL1-specific fixation effect from this result.
A whole tissue is called negative or positive from one cell population.	HPA levels describe named populations, such as glandular cells or cholangiocytes, rather than every cell in an organ (HPA: tissue IHC).	Record cell identity, compartment and intensity separately; compare like cell populations across sections (general IHC practice).

Source: UniProt O95294 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing RASAL1 staining

IHC analysis of RASAL1 using anti-RASAL1 antibody (A06423-2). RASAL1 was detected in paraffin-embedded section of rat intestine tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-RASAL1 Antibody (A06423-2) 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.

A06423-2 · IHCIHC analysis of RASAL1 using anti-RASAL1 antibody (A06423-2). RASAL1 was detected in paraffin-embedded section of rat intestine tissue. Heat

IHC analysis of RASAL1 using anti-RASAL1 antibody (A06423-2). RASAL1 was detected in paraffin-embedded section of mouse spleen tissue. Heat mediated antigen retrieval was performed in citrate buffer (pH6, epitope retrieval solution) for 20 mins. The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml rabbit anti-RASAL1 Antibody (A06423-2) 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.

A06423-2 · IHCIHC analysis of RASAL1 using anti-RASAL1 antibody (A06423-2). RASAL1 was detected in paraffin-embedded section of mouse spleen tissue. Heat

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000111344-RASAL1/tissue/cerebral+cortex#imid_11290583

HPA · Parathyroid glandHuman Protein Atlas IHC, Parathyroid gland. Source: https://www.proteinatlas.org/ENSG00000111344-RASAL1/tissue/parathyroid+gland#imid_11290466

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000111344-RASAL1/tissue/colon#imid_11290493

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000111344-RASAL1/tissue/liver#imid_11290502

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041650; green = RASAL1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for RASAL1 IHC & IF

🧪Run adrenal gland first: its glandular cells should stain (HPA: adrenal glandular cells High). Use liver cholangiocytes as the negative tissue (HPA: liver cholangiocytes Not detected); compare other cells on the adrenal slide with background, but their negative status is not established by the supplied HPA row.

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: Liver (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show RASAL1 in A-431, MCF-7, U2OS, with annotated localisation: Plasma membrane (approved), Cytosol (approved) (HPA subcellular).

Technical controls: Include no-primary (secondary-only) and isotype-matched rabbit IgG controls, plus RASAL1-knockout tissue if available (caption: rabbit primary antibody). For the adrenal slide, block endogenous peroxidase and check endogenous biotin background before interpreting SABC/DAB staining (caption: SABC/DAB detection).

⚠️Feasibility: A target-specific fixation window and fixation effect are unreported; the selected A06423-2 tissue-IHC caption does not state the fixative. The reported paraffin-section method uses citrate retrieval at pH 6 for 20 minutes, but does not establish that retrieval is required under other conditions (caption: citrate retrieval). HPA has ICC-IF images in A-431, MCF-7 and U2OS cells, but the supplied evidence does not establish whether IF or frozen sections are easier than paraffin IHC; on the adrenal slide, endogenous peroxidase or biotin may complicate SABC/DAB interpretation (HPA: ICC-IF cell lines; caption: SABC/DAB detection).

HPA tissue IHC evidence for RASAL1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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 →
Esophagus	Squamous epithelial cells	High	Protein (IHC)	HPA →
Oral mucosa	Squamous epithelial cells	High	Protein (IHC)	HPA →
Parathyroid gland	Glandular cells	High	Protein (IHC)	HPA →
Salivary gland	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →
Lymph node	Germinal center cells	Not detected	Protein (IHC)	HPA →
Ovary	Ovarian stroma cells	Not detected	Protein (IHC)	HPA →
Spleen	Cells in red pulp	Not detected	Protein (IHC)	HPA →

Section 3

Advanced RASAL1 IHC Tips

These questions focus on interpreting RASAL1 staining in paraffin sections, with one follow-up question on IF/ICC.

What retrieval should I try first when RASAL1 staining is weak?

Start with heat-mediated citrate retrieval at pH 6 for 20 minutes in paraffin sections (datasheet A06423-2; selected IHC caption). Keep heating, cooling, and section handling consistent while comparing staining across runs (standard IHC practice). The selected image used 1 μg/mL primary antibody overnight at 4°C, so reproduce those conditions before attributing weak staining to retrieval (selected IHC caption). If signal remains weak, test a different retrieval buffer or heating duration on matched sections, assessing tissue integrity and background alongside signal (standard IHC practice).

Could fixation explain variable RASAL1 staining between paraffin blocks?

RASAL1-specific fixation sensitivity is unknown: the selected paraffin-section caption does not report a fixative (selected IHC caption). Document each block's fixative and processing history, and compare sections with matched thickness, retrieval, and staining conditions before assigning the difference to antigen abundance (standard IHC practice). Include a consistently processed positive control, such as adrenal gland glandular cells reported as strongly stained, in each run (HPA: High in adrenal gland glandular cells). If blocks still differ, report the variation as a possible preanalytical effect; staining alone cannot establish whether fixation altered the epitope (standard IHC practice).

Which compartments should count as plausible RASAL1 signal in chromogenic sections?

Prioritise interpretable membranous and cytoplasmic staining in morphologically intact cells (HPA tissue IHC: membranous and cytoplasmic expression). Plasma membrane and cytosol are approved main locations, with cell junctions an additional approved location in subcellular imaging (HPA subcellular). RASAL1 has no annotated transmembrane segment, so peripheral staining is plausible without treating it as evidence of membrane insertion (UniProt O95294 topology; HPA subcellular). Score nuclear-only deposits separately and check whether they persist in controls, because a nuclear location is not among the supplied supported locations (HPA subcellular; standard IHC practice).

Can this stain distinguish RASAL1 isoforms or reveal a masked epitope?

Do not assign an isoform from this stain: RASAL1 has 4 listed isoforms, while the selected tissue-IHC caption gives no antibody epitope or isoform specificity (UniProt O95294 isoforms; selected IHC caption). The protein contains 2 C2 domains, a Ras-GAP domain, and a PH domain, so epitope position matters when interpreting potential isoform coverage (UniProt O95294 domains). Obtain the immunogen sequence and compare it with the isoforms before making an isoform-specific claim (standard antibody-validation practice). If staining differs after retrieval, evaluate preservation and background on matched sections; that change alone does not locate a masked epitope (standard IHC practice).

How should I follow up an IHC pattern with multiplex IF/ICC?

Use IF/ICC as a separate follow-up assay and pair RASAL1 with a marker identifying the expected cell type, such as the glandular population being assessed in adrenal or parathyroid tissue (HPA tissue IHC: High in glandular cells). Choose a fluorophore in a channel with low measured tissue autofluorescence and include single-label controls before interpreting overlap (standard IF practice). RASAL1 is reported at plasma membrane, cytosol, and cell junctions and has no transmembrane segment (HPA subcellular; UniProt O95294 topology). For an intracellular or inner-membrane-facing epitope, titrate permeabilisation; confirm epitope accessibility before treating weak IF as disagreement with IHC (standard IF practice).

How can I separate RASAL1 staining from diffuse DAB background?

The selected paraffin-section method used 10% goat serum block, biotinylated secondary antibody, a streptavidin–biotin detection complex, and DAB (selected IHC caption). Run a no-primary control to assess secondary and detection background, and inspect endogenous biotin and peroxidase contributions when using this detection scheme (standard IHC practice). Apply a peroxidase block and optimise washes as general chromogenic IHC steps, then compare the controls with identically developed test sections (standard IHC practice). Retain a pattern only when cellular signal exceeds control deposits and fits the reported membranous or cytoplasmic distribution (HPA tissue IHC; standard IHC interpretation).

How should I quantify heterogeneous RASAL1 staining across sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and scoring threshold before reviewing groups, then record the percentage of positive viable cells and staining intensity within each compartment (standard IHC scoring practice). An H-score weights the percentages at intensity grades 0–3, yielding 0–300; report membranous and cytoplasmic scores separately when distinguishable (standard IHC H-score; HPA tissue IHC: membranous and cytoplasmic expression). For counts, express positive cells per mm² of viable tissue or per eligible cell population, using the same denominator across samples (standard IHC quantification). Compare matched staining runs and cell types, since RASAL1 expression varies by tissue and cell population (HPA tissue IHC).

When is an apparent RASAL1-positive area more likely artefact?

A convincing area contains intact cells with membranous or cytoplasmic signal above matched controls, consistent with the reported tissue pattern (HPA tissue IHC; standard IHC practice). Reconsider isolated nuclear-only staining, since supported locations are plasma membrane, cytosol, and cell junctions (HPA subcellular). Treat strong deposits at section edges, necrotic regions, or sites of endogenous enzyme activity cautiously, and compare them with no-primary and peroxidase controls (standard IHC practice). Cell identity also matters: glandular cells in adrenal gland are reported High, whereas liver cholangiocytes are Not detected; use these as context rather than absolute diagnostic rules (HPA tissue IHC).

Boster reagents

Best RASAL1 / RasGAP-activating-like protein 1 IHC Antibodies

The IHC-validated antibody has real paraffin-section data from rat intestine and mouse spleen and intestine (A06423-2 IHC captions). Human, mouse and rat reactivity is listed (catalog: A06423-2); IF data are absent (catalog: A06423-2).

Real IHC data

Anti-RASAL1/RASAL1 Antibody ®

Cat # A06423-2

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A06423-2 will render with a rat intestine paraffin-section IHC figure (A06423-2 figure caption). Its other IHC captions report mouse spleen and intestine paraffin sections (A06423-2 IHC captions); the catalog lists human, mouse and rat reactivity (catalog: A06423-2).

Which to pick: For tissue IHC, choose A06423-2, a rabbit polyclonal antibody listed for IHC, with paraffin-section images and a recommended concentration of 0.5–1 μg/ml (catalog: A06423-2; A06423-2 IHC captions). The pictured rat intestine section used citrate retrieval at pH 6 for 20 minutes; its fixative is unreported (A06423-2 figure caption). For cross-species work, the catalog lists human, mouse and rat reactivity, though the IHC images show only rat and mouse tissue; no IF/ICC application or IF image is listed, so there is no IF/ICC-supported SKU to pick (catalog: A06423-2; A06423-2 IHC captions).

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

References

UniProt Consortium. UniProt entry O95294 (RASL1_HUMAN, RasGAP-activating-like protein 1).
Human Protein Atlas. RASAL1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. RASAL1 subcellular location (ICC-IF): Mainly localized to the plasma membrane and cytosol. In addition localized to the cell junctions..
Human Protein Atlas. RASAL1 antibody validation summary (1 antibodies).
Genome-wide CRISPR/Cas9 screening identifies a targetable MEST-PURA interaction in cancer metastasis. EBioMedicine 2023 — PMC10192437.
Epigenetic Suppression of RASAL1 by HDAC3 and Cofactor YY1 Promotes Fibroblast-Myofibroblast Transition and Renal Fibrosis. Research (Washington, D.C.) 2026 — PMC12852569.
Hypermethylated RASAL1's promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs. BMC cancer 2024 — PMC11312928.
High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis. Nature communications 2018 — PMC6115451.
PubMed PMID:9751798 — UniProt-cited evidence.
PubMed PMID:16541075 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.