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CRKL / Crk-like protein · IHC design guide
Design Immunohistochemistry for CRKL
Plan chromogenic CRKL IHC in paraffin sections using colon glandular cells as a candidate positive control (HPA tissue IHC). Assess cytoplasmic staining while accounting for the uncertain agreement between antibody staining and RNA expression (HPA tissue IHC).
Evidence assembled Oct 2026 · For research use; verify linked source records and product datasheet before use
CRKL 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 several tissues (HPA tissue IHC) | |
| Staining pattern | Colon glandular cells: high cytoplasmic staining (HPA tissue IHC) | |
| Antigen retrieval | EDTA pH 8.0 HIER, heat-mediated (datasheet M02100-2) | |
| Positive control | Colon+4 more · see all | |
| Negative control | Adipose tissue+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 | Antibody staining and RNA show low consistency (HPA tissue IHC) | |
| Regulation | No expression regulator reported (UniProt) | |
| Isoform / epitope | No isoforms; one 1–303 chain with no signal peptide (UniProt) |
Section 1
Recommended CRKL IHC & IF Protocols
The catalog antibody’s IHC-P protocol (datasheet: M02100-2) is accompanied by published CRKL staining protocols for gastric cancer tissue microarrays (PMC3388458) and breast tissue microarrays (PMC6679112).
| Sample | Paraffin-embedded mouse brain tissue; fixative not specified (datasheet M02100-2) |
| Fixation | Image fixative and duration unreported (datasheet M02100-2); verify before use. |
| Sectioning | 4–5 µm sections on charged slides (standard) |
| Deparaffinisation | Xylene, graded ethanol series to water (standard) |
| Antigen retrieval | Heat retrieval: EDTA pH 8.0 (datasheet M02100-2); 20 min, 95–100 °C (standard) |
| Peroxidase block | 3% H2O2, 10 min, room temperature (standard) |
| Blocking | 10% goat serum (datasheet M02100-2) |
| Primary antibody | Rabbit monoclonal (clone 32C63) anti-CRKL, 1:50-1:200 (datasheet M02100-2) |
| Primary incubation | Overnight at 4 °C (datasheet M02100-2) |
| Detection | HRP-conjugated secondary, DAB chromogen (datasheet M02100-2) |
| Counterstain | Hematoxylin, blue, dehydrate and mount (standard) |
| Expected result | CRKL-positive staining in glandular cells of colon (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression in several tissues. No signal in the no-primary control. |
💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: M02100-2). Tris-EDTA at pH 9.0 is a published alternative for gastric cancer sections (PMC3388458).
Section 2
What Is the Expected CRKL Staining Pattern?
For paraffin-section IHC, expect predominantly cytoplasmic CRKL staining in glandular cells of colon, small intestine, stomach, rectum, fallopian tube, and seminal vesicle (HPA tissue IHC: High in these cells; cytoplasmic expression in several tissues). Treat this as a provisional pattern: HPA rates the tissue profile Uncertain because antibody staining and RNA expression have low consistency (HPA tissue IHC: Uncertain). CRKL has no transmembrane segment (UniProt P46109: topology).
| Distinct cytoplasmic staining in colon or small-intestine glandular cells, with identifiable tissue structure. | This matches the reported IHC compartment and high-staining cell populations (HPA tissue IHC: cytoplasmic expression; High in colon and small-intestine glandular cells). Compare cells within the same section and a separate lower-staining tissue; intensity alone cannot resolve the profile's Uncertain reliability (HPA tissue IHC: Uncertain). |
| A crisp, exclusively cell-surface outline replaces the expected cytoplasmic pattern. | Investigate an artefact or off-target signal: CRKL has no transmembrane segment (UniProt P46109: topology), while the reported tissue-IHC pattern is cytoplasmic (HPA tissue IHC: cytoplasmic expression). Nuclear signal needs separate interpretation because ICC-IF reports a mainly nucleoplasmic pattern (HPA subcellular: Nucleoplasm enhanced). |
| Strong signal appears in adipocytes while nearby glandular cells show little or none. | Consider cross-reactivity or endogenous detection activity after checking morphology and controls (general IHC practice). HPA reports adipocytes as Not detected in adipose tissue and breast, whereas several glandular populations are High (HPA tissue IHC: adipocytes Not detected; glandular cells High). This comparison is a warning flag, not proof of antibody failure (HPA tissue IHC: Uncertain). |
| Pale stain covers extracellular spaces, tissue edges, and multiple cell types without cell boundaries. | Treat the pattern as background until a reagent control and tissue morphology support cellular staining (general IHC practice). A widespread haze cannot be assigned to CRKL from the reported cytoplasmic cell pattern (HPA tissue IHC: cytoplasmic expression in several tissues); inspect blocking, washes, and chromogen development (general IHC practice). |
| No glandular-cell signal is visible in colon or small intestine. | Check section integrity and the assay controls before calling the specimen negative (general IHC practice). These are useful comparison tissues because HPA reports High glandular-cell staining there (HPA tissue IHC: High in colon and small intestine). Their absence may reflect assay failure or biological variation; the reference profile remains Uncertain (HPA tissue IHC: Uncertain). |
💡Expected CRKL appearanceCall the IHC result provisionally positive when glandular cells show clear, predominantly cytoplasmic staining of appreciable intensity in a reported High tissue (HPA tissue IHC: High glandular cells; cytoplasmic expression); diffuse extracellular color or isolated adipocyte staining warrants a background check (HPA tissue IHC: adipocytes Not detected; general IHC practice).
| Reference-tissue choice | Colon and small intestine offer reported High glandular-cell staining; adipose-tissue adipocytes offer a reported Not detected comparison (HPA tissue IHC: High; Not detected). Use the contrast as an assay check, while retaining HPA's Uncertain reliability rating (HPA tissue IHC: Uncertain). |
| Antibody validation | The listed antibody HPA001100 has Uncertain IHC validation; HPA000532 has Enhanced ICC validation without an IHC rating (HPA antibodies: IHC Uncertain; ICC Enhanced). Validation in one application does not establish the other's expected slide performance (general assay interpretation). |
| Protein architecture and modification | CRKL is one annotated chain, residues 1–303, with SH2 and two SH3 domains and reported phosphotyrosines at 127 and 207 (UniProt P46109: chain, domains, modified residues). The payload gives no antibody epitope, so these features cannot predict retrieval conditions or a phospho-specific staining pattern (UniProt P46109: record scope). |
| IF/ICC Q: Should the IHC cytoplasmic pattern be imposed on IF images? | A: No. HPA reports mainly nucleoplasmic ICC-IF localisation, with additional cytosol, whereas tissue IHC is described as cytoplasmic (HPA subcellular: Nucleoplasm enhanced, Cytosol approved; HPA tissue IHC: cytoplasmic). Interpret each application against its own evidence. |
| Detection background | Endogenous enzyme activity or nonspecific reagent binding can create chromogenic color unrelated to primary-antibody binding (general IHC practice). A reagent control helps identify this contribution; the HPA cell-pattern observations do not determine its source in a particular section (HPA tissue IHC: profile scope). |
| Situation | Likely cause | Next action |
|---|---|---|
| The reported High glandular cells remain blank. | The assay may not have produced a detectable primary-antibody signal, or the sampled cells may differ from the reference (general IHC practice; HPA tissue IHC: Uncertain). | Confirm glandular-cell identity and section quality, then review the IHC-validated antibody's supplied IHC-P instructions and run appropriate assay controls (general IHC practice). Compare with colon or small intestine (HPA tissue IHC: High glandular cells). |
| The whole section turns brown, including spaces between cells. | Excess detection background, incomplete washing, or endogenous enzyme activity may obscure a cellular pattern (general IHC practice). | Inspect a primary-omission control, blocking and wash steps, and chromogen development; score only resolved cellular signal (general IHC practice). |
| A sharply membranous pattern dominates. | The compartment conflicts with reported cytoplasmic tissue staining and a protein lacking a transmembrane segment (HPA tissue IHC: cytoplasmic; UniProt P46109: topology). | Check morphology and controls, then repeat with an independently validated IHC reagent if available (general IHC practice). Avoid interpreting an outline alone as CRKL localisation. |
| Adipocytes stain as strongly as expected glandular cells. | This differs from HPA's Not detected adipocyte observations and could reflect nonspecific or endogenous color (HPA tissue IHC: adipocytes Not detected; general IHC practice). | Compare a primary-omission control and inspect whether staining follows adipocyte boundaries; retain the HPA reliability caveat when judging disagreement (general IHC practice; HPA tissue IHC: Uncertain). |
| An IHC section looks nuclear rather than cytoplasmic. | The tissue-IHC reference is cytoplasmic, but ICC-IF reports mainly nucleoplasmic CRKL; the two application observations differ (HPA tissue IHC: cytoplasmic; HPA subcellular: Nucleoplasm enhanced). | Verify the section's cell types, staining controls, and antibody IHC validation before assigning the nuclear signal to CRKL (general IHC practice; HPA antibodies: HPA001100 IHC Uncertain). |
| Retrieval changes the staining pattern across runs. | Target-specific fixation effects are not established by the supplied assay evidence. Verify with a matched IHC source before attributing a result to fixation. | Document retrieval conditions and compare runs with the same reference tissue and controls; follow the IHC-validated antibody's supplied IHC-P instructions (general IHC practice). Do not infer a CRKL-specific fixation effect from the HPA pattern. |
Experiment evidence
IHC & IF images showing CRKL staining
M02100-2 · IHCIHC analysis of CRKL using anti-CRKL antibody (M02100-2). CRKL was detected in a paraffin-embedded section of mouse brain tissue. Heat media
M02100-2 · IHCIHC analysis of CRKL using anti-CRKL antibody (M02100-2). CRKL was detected in a paraffin-embedded section of mouse brain tissue. Heat media
A02100-1 · IFIF analysis of CRKL using anti-CRKL antibody (A02100-1) and anti-Tubulin Alpha antibody (M03989-3). CRKL was detected in immunocytochemical
HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000099942-CRKL/tissue/cerebral+cortex#imid_375766
HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000099942-CRKL/tissue/colon#imid_375684
HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000099942-CRKL/tissue/liver#imid_375677
HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000099942-CRKL/tissue/kidney#imid_375694
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA000532; green = CRKL, blue = nucleus, red = microtubules.
HPA · ICC-IF Rh30Human Protein Atlas immunofluorescence, Rh30, antibody HPA000532; green = CRKL, blue = nucleus, red = microtubules.
← swipe / scroll for more images →
Real IHC / IF data behind the staining-pattern expectations in this section.
Sample controls for CRKL IHC & IF
🧪Run colon first: glandular cells should stain (HPA: High in colon glandular cells). Use adipose tissue as the negative comparator, focusing on adipocytes (HPA: Not detected in adipocytes); on the colon slide, assess adjacent cells without specific staining for background-level DAB, without assuming they are CRKL-negative.
Positive control tissue: Colon (Glandular cells, HPA High)
Negative control tissue: Adipose tissue (HPA Not detected)
ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CRKL in Rh30, U2OS, HeLa BAC 5551, HeLa , with annotated localisation: Nucleoplasm (enhanced) (HPA subcellular).
Technical controls: Include no-primary (secondary-only), a rabbit isotype control matched to the primary antibody’s clonality (caption: rabbit anti-CRKL), and a CRKL-knockout biological negative. Block endogenous peroxidase before chromogenic detection and assess any staining outside the expected glandular cells separately (standard IHC practice; HPA: High in colon glandular cells).
⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected M02100-2 paraffin-section caption does not state the fixative (caption: fixative not stated). Heat retrieval in EDTA at pH 8.0 was used for the pictured mouse brain section, but retrieval dependency in colon is unreported (caption: EDTA pH 8.0, mouse brain). The supplied evidence does not establish that frozen sections or IF are easier; HPA ICC-IF images show mainly nucleoplasmic and additional cytosolic localization, which should not be treated as an IHC processing result (HPA: subcellular).
HPA tissue IHC evidence for CRKL
Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Uncertain — Low 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 |
|---|---|---|---|---|
| Colon | Glandular cells | High | Protein (IHC) | HPA → |
| Fallopian tube | Glandular cells | High | Protein (IHC) | HPA → |
| Rectum | Glandular cells | High | Protein (IHC) | HPA → |
| Seminal vesicle | Glandular cells | High | Protein (IHC) | HPA → |
| Small intestine | 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 → |
| Adrenal gland | Glandular cells | Not detected | Protein (IHC) | HPA → |
| Breast | Adipocytes | Not detected | Protein (IHC) | HPA → |
| Caudate | Glial cells | Not detected | Protein (IHC) | HPA → |
| Esophagus | Squamous epithelial cells | Not detected | Protein (IHC) | HPA → |
Section 3
Advanced CRKL IHC Tips
Troubleshoot CRKL staining in paraffin sections by checking retrieval, staining controls and cell specific localisation before comparing chromogenic signal (datasheet M02100-2; HPA tissue IHC).
What retrieval should I use if CRKL staining is weak in paraffin sections?
Begin with heat mediated EDTA retrieval at pH 8.0 for paraffin CRKL IHC (datasheet M02100-2). The selected caption reports this buffer and heat treatment but gives no heating duration, so record temperature, duration and cooling consistently during optimisation (datasheet M02100-2; standard IHC practice). Keep the catalog antibody at its reported 1:50, overnight at 4°C, while comparing retrieval conditions (datasheet M02100-2; standard IHC practice). Compare staining in matched sections with the same chromogenic detection and counterstain, watching for tissue damage or widespread nonspecific colour (standard IHC practice). Change antibody concentration only after retrieval and section integrity have been assessed (standard IHC practice).
Could fixation explain variable CRKL staining across paraffin sections?
Target specific fixation sensitivity for CRKL is unknown from the supplied evidence (datasheet M02100-2). The selected image describes a paraffin embedded mouse brain section but does not state its fixative or fixation time, so it cannot establish a preferred fixation condition (datasheet M02100-2). Record fixative, duration and processing for each specimen, then compare sections processed alike when assessing signal loss (standard IHC practice). Include a concurrently stained reference section and keep EDTA retrieval at pH 8.0 constant during that comparison (datasheet M02100-2; standard IHC practice). Do not attribute a weak result to CRKL specific fixation effects without a controlled fixation comparison (standard IHC practice).
How should I evaluate nuclear and cytoplasmic CRKL staining?
Review nuclear and cytoplasmic staining separately because tissue IHC reports cytoplasmic expression, while subcellular IF reports mainly nucleoplasm with additional cytosol (HPA tissue IHC; HPA subcellular). UniProt supplies no CRKL subcellular annotation, so neither pattern alone establishes a universal compartment rule (UniProt P46109). Check whether signal follows intact cell boundaries and persists in comparable cells across the section (standard IHC practice). Score each compartment independently under identical retrieval and chromogenic development conditions, and document the observed pattern (standard IHC practice). Treat a new nuclear dominant IHC pattern as a finding requiring controls and orthogonal support, given the reported tissue IHC and IF difference (HPA tissue IHC; HPA subcellular; standard IHC practice).
Could epitope selection account for inconsistent CRKL IHC staining?
Check the antibody's documented immunogen or mapped epitope before interpreting inconsistent paraffin staining, since the supplied caption does not identify the recognised region (datasheet M02100-2; standard IHC practice). CRKL has an SH2 domain at residues 14–102 and SH3 domains at 123–183 and 235–296 (UniProt P46109). Annotated phosphotyrosines at 127 and 207 could matter only if an antibody's actual epitope and modification sensitivity support that inference (UniProt P46109; standard IHC practice). No isoforms are listed in the supplied record, so avoid assigning different staining patterns to an undocumented isoform (UniProt P46109). Compare another validated epitope or an orthogonal assay if specificity remains uncertain (standard IHC practice).
How can IF help investigate an ambiguous chromogenic CRKL pattern?
Use IF as a separate follow up to ambiguous paraffin IHC, with its own validated conditions rather than assuming the chromogenic conditions transfer directly (standard IHC/IF practice). Multiplex CRKL with an epithelial marker when examining glandular cells, which are reported as strongly stained in several tissues (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores after checking the specimen's autofluorescence, and include single stain controls to assess bleed through (standard IF practice). CRKL has no transmembrane segment, so assess permeabilisation for intracellular epitopes while preserving cellular structure (UniProt P46109; standard IF practice). Compare nuclear and cytosolic signals with the reported IF distribution, while treating the tissue IHC pattern as separate evidence (HPA subcellular; HPA tissue IHC).
What should I check when CRKL DAB staining looks diffuse?
First compare the section with a no primary control to distinguish antibody dependent colour from detection background (standard IHC practice). The reported paraffin image used 10% goat serum blocking, a peroxidase conjugated secondary and DAB development (datasheet M02100-2). Apply an endogenous peroxidase block and inspect pigment rich or damaged areas before calling diffuse brown colour CRKL signal (standard chromogenic IHC practice). If background persists, check wash stringency, secondary compatibility and DAB development time while keeping retrieval at the reported EDTA pH 8.0 condition (datasheet M02100-2; standard IHC practice). Interpret any remaining diffuse signal against cellular morphology and control sections (standard IHC practice).
How should I quantify CRKL staining across tissue sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because tissue IHC reports cytoplasmic CRKL while subcellular IF also reports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). For chromogenic IHC, record the percentage of positive cells and staining intensity, or calculate an H score from intensity weighted percentages on a 0–300 scale (standard IHC practice). Normalise cell counts to the number of eligible intact cells, or positive cell density to measured tissue area in mm² (standard IHC practice). Use identical retrieval, development, imaging and thresholds across groups, and exclude folds and necrotic regions by a preset rule (standard IHC practice). Report nuclear and cytoplasmic scores separately if both are present (standard IHC practice).
When is a CRKL positive result convincing rather than artefactual?
A convincing result should follow intact cellular morphology, recur in comparable cells and exceed no primary control staining (standard IHC practice). Cytoplasmic signal is compatible with the reported tissue IHC profile, but that source labels its CRKL staining reliability uncertain because of low agreement with RNA expression (HPA tissue IHC). Nucleoplasmic signal has separate IF support and should be checked with compartment specific controls before interpreting it in chromogenic sections (HPA subcellular; standard IHC practice). Be cautious with staining concentrated at section edges, folds or necrosis, or colour attributable to endogenous peroxidase (standard IHC practice). Use independent target evidence when a strong result conflicts with expected cellular context, and document the exact population scored (standard IHC practice).
Boster reagents
Best CRKL / Crk-like protein IHC Antibodies
Anti-CRKL antibodies have illustrated paraffin-section IHC in mouse brain (M02100-2 image caption) and IF/ICC in U87 cells (A02100-1 image caption).
Real IHC data
Anti-CrkL Rabbit Monoclonal Antibody
Cat # M02100-2
Real IF data
Anti-CRKL Antibody ®
Cat # A02100-1
M02100-2 renders with IHC in a paraffin-embedded mouse brain section using EDTA retrieval at pH 8.0 and 1:50 primary antibody (M02100-2 image caption). A02100-1 renders with IF/ICC in U87 cells using 5 μg/mL primary antibody (A02100-1 image caption).
Which to pick: Choose M02100-2 for tissue IHC: it is a rabbit monoclonal listed for IHC (catalog), and its own caption documents a paraffin-embedded section; the fixative is unreported (M02100-2 image caption). Choose A02100-1 for IF/ICC in human samples: IF and ICC are listed applications with human reactivity (catalog), and its image shows U87 cells (A02100-1 image caption). For work across human, mouse, and rat, M02100-2 lists reactivity with all three species (catalog), with paraffin-section IHC images in mouse and rat brain (M02100-2 image captions).
Each figure is that product's own IHC / IF validation image from its datasheet.
References
- UniProt Consortium. UniProt entry P46109 (CRKL_HUMAN, Crk-like protein).
- Human Protein Atlas. CRKL tissue IHC expression (reliability: Uncertain).
- Human Protein Atlas. CRKL subcellular location (ICC-IF): Mainly localized to the nucleoplasm. In addition localized to the cytosol..
- Human Protein Atlas. CRKL antibody validation summary (2 antibodies).
- The CRKL gene encoding an adaptor protein is amplified, overexpressed, and a possible therapeutic target in gastric cancer. Journal of translational medicine 2012 — PMC3388458.
- High CC chemokine receptor 7 expression improves postoperative prognosis of lung adenocarcinoma patients. British journal of cancer 2013 — PMC3778295.
- Increased Soluble CrkL in Serum of Breast Cancer Patients Is Associated with Advanced Disease. Cancers 2019 — PMC6679112.
- SLC7A5 Functions as a Downstream Target Modulated by CRKL in Metastasis Process of Gastric Cancer SGC-7901 Cells. PloS one 2016 — PMC5112787.
- PubMed PMID:8361759 — UniProt-cited evidence.
- PubMed PMID:15461802 — UniProt-cited evidence.
- PubMed PMID:14702039 — UniProt-cited evidence.
Immunohistochemistry protocol sheet for CRKL (Crk-like protein). Expected localisation Cytoplasmic staining in several tissues (HPA tissue IHC). Antigen retrieval: EDTA pH 8.0 HIER, heat-mediated (datasheet M02100-2). Validated IHC antibody: M02100-2 at 1:50-1:200 (the product's own IHC validation image is shown). Controls (HPA): positive Colon (Glandular cells); negative Adipose tissue. IHC-P workflow on paraffin sections; fixative unspecified. Protocol steps: 1. Sample: Paraffin-embedded mouse brain tissue; fixative not specified (datasheet M02100-2); 2. Fixation: Image fixative and duration unreported (datasheet M02100-2); 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 retrieval: EDTA pH 8.0 (datasheet M02100-2); 20 min, 95–100 °C (standard); 6. Peroxidase block: 3% H2O2, 10 min , room temperature (standard); 7. Blocking: 10% goat serum (datasheet M02100-2); 8. Primary antibody: Rabbit monoclonal (clone 32C63) anti-CRKL, 1:50-1:200 (datasheet M02100-2); 9. Primary incubation: Overnight at 4 °C (datasheet M02100-2). Watch for: Fixation: Keep fixation consistent across sections (standard IHC practice; not target-specific); Caveat: Antibody staining and RNA show low consistency (HPA tissue IHC); Isoform / epitope: No isoforms; one 1–303 chain with no signal peptide (UniProt); Regulation: No expression regulator reported (UniProt). Evidence: UniProt P46109, HPA tissue IHC · Uncertain, HPA ICC-IF · Mainly localized to the nucleoplasm. In addition localized to the…, Datasheet M02100-2, PMC3388458, PMC6679112. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRKL/immunohistochemistry-ihc-guide.