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CRKL / Crk-like protein · IHC design guide
Design Immunohistochemistry for CRKL
Plan CRKL staining in paraffin sections with the IHC-validated antibody at 1:50–1:200 (datasheet: M02100-2). Assess cytoplasmic staining in glandular cells while accounting for the uncertain tissue-IHC reliability (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 | Cytoplasmic staining in glandular cells (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 | Staining has low consistency with RNA expression (HPA tissue IHC) | |
| Regulation | Expression regulation not annotated (UniProt) | |
| Isoform / epitope | 0 isoforms; no processing variants annotated (UniProt) |
Section 1
Recommended CRKL IHC & IF Protocols
The catalog antibody uses EDTA pH 8.0 retrieval (datasheet M02100-2). Four published IHC protocols describe gastric, lung and breast tissue sections (PMC3388458; PMC3778295; PMC6679112; PMC5112787).
| 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 EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet M02100-2); a published gastric tissue protocol used Tris-EDTA pH 9.0 (PMC3388458).
Section 2
What Is the Expected CRKL Staining Pattern?
In paraffin-section IHC, expect predominantly cytoplasmic CRKL staining in glandular cells of colon, fallopian tube, rectum, seminal vesicle, small intestine, and stomach (HPA tissue IHC: High; cytoplasmic expression in several tissues). CRKL has no transmembrane segment (UniProt P46109 topology). Treat this as a provisional pattern: HPA rates its tissue IHC reliability Uncertain because staining and RNA expression show low consistency (HPA tissue IHC: Uncertain).
| Cytoplasmic staining in glandular cells of colon or small intestine. | This matches a reported high-staining cell type and compartment (HPA tissue IHC: High; cytoplasmic expression). Score the glandular cells themselves, then compare staining with a negative control. The match supports interpretation but cannot establish antibody specificity alone because the tissue IHC profile is rated Uncertain (HPA tissue IHC: reliability). |
| Predominantly nuclear staining with little cytoplasmic staining in paraffin-section IHC. | This differs from the reported tissue IHC pattern (HPA tissue IHC: cytoplasmic expression). Check whether the apparent nuclear signal also occurs in a negative control, and review counterstain and detection background (general IHC practice). Nuclear staining is not inherently impossible: HPA reports nucleoplasm as the main location in ICC-IF, an observation from a different application (HPA subcellular: nucleoplasm, enhanced). |
| Strong staining in adipocytes or esophageal squamous epithelial cells. | Those cell types are reported as Not detected in the cited tissue IHC profile (HPA tissue IHC: adipose tissue; esophagus). Investigate cross-reactivity and endogenous chromogenic activity with appropriate negative controls (general IHC practice). Because the HPA profile is Uncertain, discordance is a reason to investigate, not proof that an individual stained cell is false (HPA tissue IHC: reliability). |
| Diffuse color covers cells and surrounding section without a clear cellular pattern. | A diffuse deposit cannot be confidently scored as CRKL localisation. Examine negative controls and distinguish localized stain from nonspecific background or endogenous detection activity (general chromogenic IHC practice). The expected tissue IHC observation is cellular, predominantly cytoplasmic expression, rather than uniform color across the section (HPA tissue IHC: profile). |
| No glandular-cell signal in colon despite an otherwise readable section. | Colon glandular cells are reported High, so absence warrants a run-quality check (HPA tissue IHC: colon). Confirm that the IHC-validated antibody and detection system work on a known-positive control; review retrieval and primary-antibody steps using the validated procedure (general IHC practice). A single negative result does not overturn an Uncertain tissue profile (HPA tissue IHC: reliability). |
💡Expected CRKL appearanceCall a result provisionally positive when glandular cells show clear cytoplasmic chromogenic staining, especially in tissues reported High, while negative controls remain clear; widespread extracellular color or strong signal in reported Not detected cell types raises concern for a false positive (HPA tissue IHC: profile, High and Not detected; general IHC practice).
| Tissue and cell-type context | Colon, fallopian tube, rectum, seminal vesicle, small intestine, and stomach glandular cells are reported High; bronchial respiratory epithelial cells are Medium (HPA tissue IHC). Use the named cell type when interpreting a section, since an organ-level positive label does not describe every cell in that organ (general IHC interpretation). |
| Evidence strength | The tissue IHC profile is Uncertain because antibody staining and RNA expression have low consistency (HPA tissue IHC: reliability). The listed IHC antibody HPA001100 is also marked Uncertain, whereas HPA000532 is marked Enhanced for ICC, a separate application (HPA antibodies). Keep these validation labels attached to their respective assays. |
| Topology and protein forms | CRKL has no transmembrane segment, signal peptide, or propeptide, and its annotated chain spans residues 1–303 (UniProt P46109 topology and processing). These annotations offer no basis for expecting a membrane-restricted or shed-protein pattern; they do not independently establish the observed tissue compartment (UniProt P46109; HPA tissue IHC: cytoplasmic expression). |
| Phosphorylation and epitope interpretation | UniProt lists phosphotyrosines at residues 127 and 207 (UniProt P46109 modified residues). The payload gives no antibody epitope or phospho-specific validation, so a stain cannot be read as phosphorylation status or used to infer that either modification changes antigen retrieval (UniProt P46109; supplied HPA antibody data). |
| IF/ICC Q&A: should the same compartment be expected? | HPA ICC-IF reports mainly nucleoplasmic CRKL, with additional cytosolic localisation (HPA subcellular: nucleoplasm enhanced; cytosol approved). This explains why an IF image may differ from the cytoplasmic tissue IHC profile (HPA tissue IHC: profile). Interpret each application with its own evidence; the ICC validation label does not validate a paraffin-section IHC result (HPA antibodies). |
| Situation | Likely cause | Next action |
|---|---|---|
| No signal in colon glandular cells. | A reported High cell type is negative (HPA tissue IHC: colon); a failed staining run or an unsupported biological difference remains possible. | Check a known-positive section, primary-antibody application, retrieval, and detection controls against the validated IHC procedure (general IHC practice). Record the discordance without calling colon universally negative because HPA rates the profile Uncertain (HPA tissue IHC: reliability). |
| Color appears mainly in nuclei on a paraffin section. | The distribution conflicts with the cytoplasmic tissue IHC profile, although ICC-IF reports a nucleoplasmic main location (HPA tissue IHC: profile; HPA subcellular). | Inspect negative controls and cellular boundaries, then document the nuclear pattern separately from the tissue IHC expectation (general IHC interpretation). Do not transfer the ICC-IF localisation or its Enhanced antibody label directly to IHC (HPA subcellular; HPA antibodies). |
| Adipocytes or esophageal squamous epithelial cells stain strongly. | Both cell types are reported Not detected in tissue IHC (HPA tissue IHC: adipose tissue; esophagus); cross-reactivity or endogenous detection activity may contribute (general IHC practice). | Compare with a negative control and assess whether color follows cells or background (general chromogenic IHC practice). Treat disagreement as a specificity question because the reference tissue profile is Uncertain (HPA tissue IHC: reliability). |
| Diffuse stain obscures glandular-cell boundaries. | Background or overdeveloped chromogen can prevent reliable localisation (general chromogenic IHC practice). | Review negative controls, blocking, detection, and development conditions using the validated procedure; score only interpretable cells (general IHC practice). A uniform deposit does not reproduce HPA's cytoplasmic cellular pattern (HPA tissue IHC: profile). |
| A reported High tissue and a reported Not detected cell type look equally stained. | The expected cell-type contrast is absent (HPA tissue IHC: High and Not detected entries); nonspecific signal or assay variation may explain it (general IHC practice). | Compare matched controls and cell-level staining before assigning positivity (general IHC interpretation). Preserve the HPA uncertainty when reporting the result, since its tissue IHC staining and RNA data have low consistency (HPA tissue IHC: reliability). |
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 and look for staining in its glandular cells (HPA: colon glandular cells High); use adipose tissue as the negative comparator and score its adipocytes, which are listed as not detected (HPA: adipose adipocytes Not detected). On the colon slide, cells without specific staining should retain only the counterstain, with no DAB deposit; the supplied HPA row does not identify a particular colon cell type as an internal negative (HPA: colon glandular cells High; standard IHC practice).
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) and rabbit IgG controls matched to the primary antibody’s format, and, if available, a CRKL knockout specimen or immunizing-peptide block as a biological specificity control (caption: rabbit anti-CRKL; standard IHC practice). Quench endogenous peroxidase for HRP/DAB detection and inspect colonic mucus for nonspecific background (caption: HRP/DAB; standard IHC practice).
⚠️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 is a documented starting condition, but retrieval dependency has not been established (caption: EDTA pH 8.0 retrieval). The supplied evidence does not show that frozen sections or IF are easier than paraffin IHC; check colonic mucus for background when interpreting glandular staining (caption: paraffin-section IHC; HPA: ICC-IF images; standard IHC practice).
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
Use the catalog antibody’s paraffin section result as a starting point, then assess compartment, cell type and controls before scoring CRKL staining.
Which retrieval conditions should I start with for CRKL chromogenic IHC?
Start with heat mediated retrieval in EDTA at pH 8.0 (datasheet M02100-2). This is the condition used for the catalog antibody’s paraffin embedded mouse brain section (datasheet M02100-2); the caption does not report a heating time or fixative (datasheet M02100-2). Keep section thickness, heating and cooling consistent across the comparison set, then assess signal against a no primary control (standard IHC practice). If staining is weak, adjust heating duration in a small pilot before changing buffer, because retrieval can alter morphology and background (standard IHC practice). Record the tested conditions alongside the final images (standard IHC practice).
How should I troubleshoot weak CRKL staining when fixation history is uncertain?
Target specific fixation sensitivity is unknown from the supplied evidence (datasheet M02100-2). The catalog image describes a paraffin embedded section but does not state its fixative or fixation duration (datasheet M02100-2). Document the available processing history and compare sections with matched processing where possible (standard IHC practice). If weak staining tracks with a particular processing batch, test retrieval and antibody incubation on adjacent sections while checking morphology and background (standard IHC practice). Do not assign the difference to a CRKL specific fixation effect without a controlled comparison; include a no primary control to assess detection background (standard IHC practice).
Should CRKL staining in paraffin sections be cytoplasmic or nuclear?
Expect predominantly cytoplasmic staining across several tissues in the HPA tissue IHC profile, but treat that pattern cautiously because its reliability is uncertain (HPA tissue IHC). Cell based imaging instead reports mainly nucleoplasmic CRKL with additional cytosolic localisation (HPA subcellular). UniProt supplies no subcellular annotation for CRKL (UniProt P46109). Score nuclear and cytoplasmic signal separately, using the counterstain to define compartments and a no primary section to assess background (standard IHC practice). A nuclear result therefore deserves validation, rather than automatic rejection; compare its cell type and reproducibility across sections before interpreting it (HPA subcellular; standard IHC practice).
Could epitope location or phosphorylation explain inconsistent CRKL staining?
The supplied record lists 0 isoforms, so an isoform explanation is unsupported here (UniProt P46109). CRKL contains an SH2 domain at residues 14–102 and SH3 domains at 123–183 and 235–296 (UniProt P46109). Reported phosphotyrosines include residues 127 and 207 (UniProt P46109). The catalog caption does not identify the antibody epitope or establish phosphorylation dependence (datasheet M02100-2). If staining varies across processing conditions, compare matched sections and consult epitope documentation before assigning a molecular cause; retrieval can affect antibody access (standard IHC practice).
How can I use IF to check a puzzling CRKL IHC pattern?
Use IF as a separate localisation check, since HPA cell images report nucleoplasmic and cytosolic CRKL (HPA subcellular). Multiplex CRKL with a validated marker for the cell type being assessed, and inspect single channels before interpreting overlap (standard IF practice). Select a fluorophore whose emission avoids the tissue’s strongest autofluorescence, using an unstained section to identify that background (standard IF practice). CRKL has no transmembrane segment, while its reported compartments are intracellular; permeabilisation is therefore relevant for antibody access, especially if the epitope is nuclear (UniProt P46109; HPA subcellular; standard IF practice). Optimise detergent exposure against morphology and background because this antibody’s IF epitope accessibility is not supplied (standard IF practice).
What should I adjust when CRKL DAB staining is diffuse or widespread?
First compare the stained section with a no primary control to distinguish primary antibody signal from detection background (standard IHC practice). The catalog example used 10% goat serum blocking, a 1:50 primary dilution overnight at 4°C, and peroxidase detection with DAB (datasheet M02100-2). If both sections develop color, review endogenous peroxidase blocking, secondary reagent binding and DAB development time (standard IHC practice). If only the primary stained section is diffuse, titrate primary antibody and review washing while preserving a matched reference section (standard IHC practice). Judge improvement by cell boundaries and compartment definition, rather than overall darkness alone (standard IHC practice).
How should I quantify CRKL staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION
Define the cell population and compartment before scoring, because HPA tissue IHC describes cytoplasmic expression while cell imaging also reports nucleoplasmic localisation (HPA tissue IHC; HPA subcellular). For a cellular readout, report the percentage of positive cells and an H-score from 0–300 using intensity grades 0–3 (standard IHC practice). For spatial counts, report positive cells per mm² of viable tissue (standard IHC practice). Normalise counts to viable area, or positive cell counts to all evaluable cells of the same type; apply one threshold and exposure independent scoring rule across sections (standard IHC practice). Exclude folds and necrotic regions consistently (standard IHC practice).
How can I distinguish genuine CRKL signal from staining artefact?
Check whether staining follows identifiable cells and reproducible compartments rather than tissue edges, folds or necrotic regions (standard IHC practice). Cytoplasmic tissue staining has been reported, while cell imaging also supports nucleoplasmic and cytosolic localisation; a nuclear signal alone is not decisive (HPA tissue IHC; HPA subcellular). High glandular cell staining is reported in colon, fallopian tube, rectum, seminal vesicle, small intestine and stomach, but the HPA tissue result has uncertain reliability (HPA tissue IHC). Compare expected and less reactive cell populations within the same section, then inspect no primary controls for endogenous enzyme or detection signal (HPA tissue IHC; standard IHC practice). Require reproducibility before calling an unusual compartment positive (standard IHC practice).
Boster reagents
Best CRKL / Crk-like protein IHC Antibodies
Anti-CRKL antibodies have IHC images from paraffin-embedded mouse and rat brain sections (M02100-2 image captions) and an IF/ICC image from human 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 has IHC images from paraffin-embedded mouse and rat brain sections and lists human, mouse and rat reactivity (M02100-2 image captions; catalog reactivity). A02100-1 has an IF/ICC image from U87 cells and lists human reactivity (A02100-1 image caption; catalog reactivity).
Which to pick: Choose M02100-2 for tissue IHC: it is a rabbit monoclonal listed for IHC, with paraffin-section images from mouse and rat brain (catalog: clone 32C63; M02100-2 image captions). It also lists human, mouse and rat reactivity for cross-species planning, although its supplied IHC images show mouse and rat tissue (catalog reactivity; M02100-2 image captions). Choose A02100-1 for human-cell IF/ICC because its image shows CRKL staining in U87 cells; the fixative is unreported in both products’ image captions (A02100-1 image caption; 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: Staining has low consistency with RNA expression (HPA tissue IHC); Isoform / epitope: 0 isoforms; no processing variants annotated (UniProt); Regulation: Expression regulation not annotated (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, PMC3778295. QR code links to https://www.bosterbio.com/bosterbio-gene-info-cards/CRKL/immunohistochemistry-ihc-guide.