WEE1 / Wee1-like protein kinase · IHC design guide

Design Immunohistochemistry for WEE1

Plan chromogenic WEE1 IHC on paraffin sections using the catalog antibody at 0.5–1 μg/mL (datasheet A01319-1). Compare staining with the reported nuclear tissue pattern (HPA tissue IHC), and consider the decrease in WEE1 protein at M/G1 when scoring intensity (UniProt).

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

Immunohistochemistry protocol sheet for WEE1 (IHC for WEE1): expected localisation Nuclear staining in several tissues (HPA tissue IHC), antibody A01319-1, validated IHC image, and IHC protocol steps — Printable **WEE1** IHC protocol sheet — expected localisation Nuclear staining in several tissues (HPA tissue IHC), antibody A01319-1, controls and protocol steps. Open the full WEE1 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

WEE1 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	Nuclear staining in several tissues (HPA tissue IHC)
	Staining pattern	Nuclear staining in gallbladder glandular cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A01319-1)
	Positive control ⓘ	Gallbladder+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 tissue fixation consistent (standard IHC practice; not target-specific); Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image A01319-1)
	Caveat	Protein loss at M/G1 may reduce staining intensity (UniProt)
	Regulation	Protein level decreases at M/G1 (UniProt)
	Isoform / epitope	2 isoforms; epitope coverage is unreported (UniProt)

Section 1

Recommended WEE1 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet: A01319-1) with four published WEE1 IHC protocols on tissue sections (PMC2663812; PMC3751528; PMC9588842; PMC6090987).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human mammary cancer tissue; fixative not specified (datasheet A01319-1)
Fixation	Image fixative and duration unreported (datasheet A01319-1); 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 A01319-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A01319-1)
Primary antibody	Rabbit anti-WEE1, 0.5-1μg/ml (datasheet A01319-1)
Primary incubation	Overnight at 4 °C (datasheet A01319-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A01319-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	WEE1-positive staining in glandular cells of gallbladder (HPA tissue IHC: High). HPA tissue profile: Nuclear 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: A01319-1); use each published retrieval condition with its corresponding antibody protocol (PMC methods).

Section 2

What Is the Expected WEE1 Staining Pattern?

WEE1 is a nuclear protein without a transmembrane segment (UniProt P30291: subcellular location and topology). In paraffin sections, expect nuclear staining in selected cells, including gallbladder glandular cells, nasopharyngeal respiratory epithelial cells, placental decidual cells and testicular preleptotene spermatocytes (HPA tissue IHC: High). HPA calls the tissue IHC profile Approved, with medium consistency between staining and RNA expression (HPA tissue IHC: reliability).

What am I looking at on my slide?
Distinct nuclear chromogen in gallbladder glandular cells or testicular preleptotene spermatocytes, with identifiable unstained areas.	This fits the expected compartment and examples scored High (UniProt P30291: Nucleus; HPA tissue IHC: High). Assess the named cell population, since an entire section need not stain uniformly (general IHC practice).
Signal is predominantly membranous or diffuse across cytoplasm, without a convincing nuclear pattern.	Treat this as a localisation mismatch rather than evidence for WEE1 distribution: UniProt places WEE1 in the nucleus and reports no transmembrane segment (UniProt P30291: subcellular location and topology). Review controls and staining conditions (general IHC practice).
Strong staining appears in adipocytes or cardiomyocytes while the expected positive cell population is weak.	HPA reports WEE1 as Not detected in those named cells (HPA tissue IHC: adipocytes; cardiomyocytes). Investigate antibody cross-reactivity or detection artefact; an unexpected cell population alone cannot identify which cause applies (general IHC practice).
Broad chromogen covers nuclei, cytoplasm and tissue spaces, obscuring cell boundaries.	This is difficult to score as specific nuclear staining (UniProt P30291: Nucleus). Diffuse background can arise from nonspecific reagent binding or detection activity; compare reagent controls and tissue morphology before assigning WEE1 positivity (general IHC practice).
No detectable nuclear signal in a section containing gallbladder glandular cells or placental decidual cells.	Those cells are scored High in HPA tissue IHC (HPA tissue IHC: gallbladder; placenta). First check that the expected cells are present, then assess assay controls and the IHC-validated antibody; a negative result by itself does not establish absent WEE1 (general IHC practice).

💡Expected WEE1 appearanceCall an IHC result positive when distinct nuclear chromogen is visible in the relevant cells, especially populations scored High (UniProt P30291: Nucleus; HPA tissue IHC: High); broad cytoplasmic or membranous colour is a localisation warning (UniProt P30291: subcellular location and topology).

How each factor affects the staining
Tissue and cell selection	HPA scores several named populations High, while adipocytes and cardiomyocytes are Not detected (HPA tissue IHC: tissue profile). Select and score the specified cells rather than assuming every cell in a tissue shares one level (general IHC practice).
IHC validation strength	HPA marks the tissue profile Approved with medium staining–RNA consistency; the IHC listing for CAB004619 is Approved (HPA tissue IHC: reliability; HPA antibodies: CAB004619). These labels support cautious interpretation, not a claim that every staining pattern is independently confirmed.
Cell cycle context	WEE1 activity rises during S and G2 and falls at M phase; its protein level also decreases around M/G1 (UniProt P30291: function). This biological variation may inform interpretation, but these annotations do not predict an IHC score for an individual cell.
Isoforms and processing	UniProt lists two isoforms, no signal peptide or propeptide, and one annotated chain spanning residues 1–646 (UniProt P30291: isoforms and processing). The payload gives no antibody epitope, so isoform detection and epitope accessibility cannot be inferred.
IF/ICC localisation Q&A	Where might IF/ICC place WEE1? HPA reports supported nucleolar localisation in its ICC-IF data, while UniProt gives the broader nuclear location (HPA subcellular: Nucleoli; UniProt P30291: Nucleus). This is localisation context for IF/ICC, not an IHC-P protocol or a requirement that chromogenic IHC resolve nucleoli.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A High reference cell population has no nuclear chromogen.	The expected cells may be absent from the examined area, or the staining run may have failed (HPA tissue IHC: High; general IHC practice).	Confirm the named cells on the section; compare a known-positive section and assay controls, then review the IHC-validated antibody and detection run (general IHC practice).
Colour is mainly cytoplasmic or outlines cell membranes.	That distribution conflicts with annotated nuclear WEE1 and its lack of a transmembrane segment (UniProt P30291: location and topology).	Treat the signal as unconfirmed; inspect a positive tissue and reagent controls before scoring nuclear WEE1 (general IHC practice).
Adipocytes or cardiomyocytes stain strongly.	These named populations are scored Not detected, so cross-reactivity or detection artefact warrants investigation (HPA tissue IHC: adipocytes; cardiomyocytes; general IHC practice).	Compare the unexpected cells with a High reference population and appropriate reagent controls; report the discrepancy if it persists (HPA tissue IHC: High; general IHC practice).
Brown colour is widespread, including areas without clear cellular localisation.	Nonspecific binding or endogenous detection activity can obscure a nuclear pattern (general IHC practice; UniProt P30291: Nucleus).	Review blocking and detection controls, and score only interpretable cells with resolved nuclei (general IHC practice).
A low-scored population is faint while a reference population stains.	Low staining is reported in cerebral cortex neuronal cells and lung macrophages (HPA tissue IHC: Low).	Interpret those named cells against their HPA level; preserve the reference result and avoid calling faint signal an assay failure solely on intensity (HPA tissue IHC: Low; general IHC practice).
IF/ICC shows nucleolar enrichment that seems different from IHC nuclear staining.	The assays provide different localisation detail: HPA supports nucleoli by ICC-IF, while its tissue IHC profile describes nuclear expression (HPA subcellular: Nucleoli; HPA tissue IHC: profile).	Record the assay and compartment separately; evaluate chromogenic IHC by its nuclear pattern and use the IF/ICC guide for that assay (HPA tissue IHC: profile; HPA subcellular: Nucleoli).

Source: UniProt P30291 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing WEE1 staining

IHC analysis of WEE1 using anti-WEE1 antibody (A01319-1). WEE1 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-WEE1 Antibody (A01319-1) 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.

A01319-1 · IHCIHC analysis of WEE1 using anti-WEE1 antibody (A01319-1). WEE1 was detected in paraffin-embedded section of human mammary cancer tissue. Hea

IF analysis of WEE1 using anti-WEE1 antibody (A01319-1). WEE1 was detected in immunocytochemical section of U20S 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-WEE1 Antibody (A01319-1) 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.

A01319-1 · IFIF analysis of WEE1 using anti-WEE1 antibody (A01319-1). WEE1 was detected in immunocytochemical section of U20S cells. Enzyme antigen retri

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000166483-WEE1/tissue/cerebral+cortex#imid_1529600

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000166483-WEE1/tissue/colon#imid_1529518

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000166483-WEE1/tissue/liver#imid_1529535

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000166483-WEE1/tissue/kidney#imid_1529520

HPA · ICC-IF RT-4Human Protein Atlas immunofluorescence, RT-4, antibody HPA068845; green = WEE1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for WEE1 IHC & IF

🧪Run gallbladder first: its glandular cells should stain (HPA: High in gallbladder glandular cells). Use adipose tissue adipocytes as the negative tissue (HPA: Not detected in adipocytes); on the gallbladder slide, use non-glandular cells lacking nuclear signal as background comparators without assuming they are WEE1-negative (HPA: High is assigned to glandular cells).

Positive control tissue: Gallbladder (Glandular cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show WEE1 in RT-4, Rh30, U2OS, with annotated localisation: Nucleoli (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control, a host- and class-matched rabbit IgG isotype control, and WEE1-knockout tissue as a biological negative (caption: rabbit anti-WEE1 antibody; standard IHC controls). For the biotin-based DAB detection shown in the caption, block endogenous peroxidase and assess endogenous biotin background (caption: SABC with DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected caption does not state the fixative (caption: fixative not stated). The documented paraffin-section workflow uses heat retrieval in EDTA at pH 8.0, but the evidence does not establish that WEE1 staining requires that retrieval condition or that frozen sections or IF are easier (caption: EDTA retrieval; HPA: ICC-IF images available). In gallbladder, distinguish any luminal bile pigment from nuclear DAB staining (HPA: gallbladder glandular cells High; standard IHC interpretation).

HPA tissue IHC evidence for WEE1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Medium 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
Gallbladder	Glandular cells	High	Protein (IHC)	HPA →
Nasopharynx	Respiratory epithelial cells	High	Protein (IHC)	HPA →
Placenta	Decidual cells	High	Protein (IHC)	HPA →
Testis	Preleptotene spermatocytes	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Kidney	Cells in glomeruli	Not detected	Protein (IHC)	HPA →
Liver	Cholangiocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced WEE1 IHC Tips

Troubleshoot WEE1 staining in paraffin sections by checking retrieval, nuclear localisation, controls and scoring before interpreting chromogenic signal.

What retrieval should I try first when WEE1 staining is weak in paraffin sections?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 for this catalog antibody (datasheet A01319-1). The selected paraffin-section image used that retrieval before overnight incubation at 4°C with 1 μg/mL antibody (caption A01319-1). If staining is weak, vary heating duration and cooling consistently across test sections, then compare nuclear signal with background (standard IHC practice; UniProt P30291 localisation). Include a no-primary control and a positive tissue control in the same run so an apparent improvement is not simply stronger nonspecific DAB deposition (standard IHC practice; HPA tissue IHC: high gallbladder glandular-cell expression).

How can I assess whether fixation is causing weak WEE1 staining?

The selected paraffin-section caption does not report its fixative, so WEE1 sensitivity to a particular fixative or fixation duration is unknown (caption A01319-1). Record the fixative and time for each specimen, and compare sections processed with matched retrieval and detection conditions (standard IHC practice). If weak nuclear staining tracks a processing batch, review fixation, section age and retrieval together before changing antibody concentration (standard IHC practice; UniProt P30291 localisation). Use a concurrently processed positive control to distinguish a batch problem from low expression in the test cells (standard IHC practice; HPA tissue IHC: high expression in gallbladder glandular cells).

Should WEE1 staining be nuclear, nucleolar or cytoplasmic?

Prioritise nuclear staining when reading chromogenic WEE1 sections, because WEE1 is annotated as nuclear and tissue IHC shows nuclear expression in several tissues (UniProt P30291 localisation; HPA tissue IHC). Supported nucleolar localisation in ICC/IF can guide a closer look, but it does not establish that nucleoli must resolve in a DAB section (HPA subcellular: nucleoli supported; standard IHC practice). Check the haematoxylin counterstain to assign DAB to nuclei in the intended cell population (standard IHC practice). Predominantly diffuse cytoplasmic colour warrants review of background and detection controls before it is scored as WEE1 (UniProt P30291 localisation; standard IHC practice).

Could isoforms or phosphorylation explain inconsistent WEE1 staining?

WEE1 has 2 annotated isoforms, but the supplied evidence does not map this antibody’s epitope to either isoform (UniProt P30291 isoforms; caption A01319-1). Its kinase domain spans residues 299–569, and multiple modified residues include phosphoserines near residues 53, 78, 85, 123 and 127 (UniProt P30291 domains and modified residues). Epitope accessibility could vary with processing or modification, but these annotations alone do not establish a cause of weak IHC signal (standard IHC practice; UniProt P30291 modified residues). Compare retrieval conditions on matched sections and seek antibody epitope information before claiming isoform-specific or phosphorylation-dependent staining (standard IHC practice).

How should I adapt a WEE1 staining question for multiplex IF?

Use the separate IF/ICC workflow to test nuclear WEE1 signal and multiplex it with a marker identifying the cell population under study (UniProt P30291 localisation; standard IF practice). HPA reports supported nucleolar localisation in ICC/IF images from RT-4, Rh30 and U2OS cells, which provides an IF localisation check (HPA subcellular). Select spectrally separated fluorophores and place the weaker signal in a channel with low specimen autofluorescence (standard IF practice). Because WEE1 has no transmembrane segment and is nuclear, permeabilise fixed cells to permit antibody access, then optimise detergent exposure for the antibody and specimen (UniProt P30291 topology and localisation; standard IF practice).

What should I check when DAB obscures WEE1 nuclear staining?

Run a no-primary control to identify signal from the secondary and detection system, and inspect tissue edges and damaged areas for nonspecific DAB (standard IHC practice). Block endogenous peroxidase before DAB development, then check that washing and serum blocking are consistent between sections (standard IHC practice). The selected image used 10% goat serum, a biotinylated secondary and streptavidin–biotin detection, so assess background from that detection sequence when reproducing it (caption A01319-1). Titrate primary antibody around the caption’s 1 μg/mL condition while retaining a positive control; accept a change only if nuclear contrast improves (caption A01319-1; UniProt P30291 localisation; standard IHC practice).

How should I score heterogeneous WEE1 staining in paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and nuclear scoring rule before examining outcomes, because tissue IHC describes WEE1 expression by cell type (HPA tissue IHC; standard IHC practice). Report the percentage of positive nuclei and, when intensity is reproducible, an H-score from 0–300 using negative, weak, moderate and strong categories (standard IHC practice). Alternatively, count positive nuclei per mm² within a prespecified viable area and normalise to all evaluable nuclei or the defined cell population (standard IHC practice). Apply one DAB threshold and exclude folds, edges and necrotic regions consistently across slides (standard IHC practice).

How can I distinguish true WEE1 positivity from staining artefact?

Give greatest weight to signal assigned to intact nuclei in the intended cells, consistent with WEE1’s nuclear annotation and the tissue IHC profile (UniProt P30291 localisation; HPA tissue IHC). HPA reports high staining in gallbladder glandular cells and no detected staining in adipocytes, but its tissue profile has only medium consistency with RNA data (HPA tissue IHC). Treat uniform edge staining, necrotic deposits and colour also present in a no-primary control as artefact candidates (standard IHC practice). Check endogenous peroxidase blocking and compare viable interior fields before interpreting DAB intensity as expression; staining alone does not measure WEE1 kinase activity (standard IHC practice; UniProt P30291 function).

Boster reagents

Best WEE1 / Wee1-like protein kinase IHC Antibodies

A01319-1 has IHC data from paraffin-embedded human mammary cancer tissue and IF data from U20S cells (catalog image captions); listed reactivity covers human, mouse, and rat (catalog).

Real IHC data

Anti-WEE1 Antibody ®

Cat # A01319-1

View product →

A01319-1 is shown in IHC on paraffin-embedded human mammary cancer tissue (catalog IHC caption). A01319-1 is shown in IF/ICC on U20S cells (catalog IF caption).

Which to pick: For tissue IHC, choose A01319-1: its image caption documents a paraffin section, EDTA pH 8 retrieval, and 1 μg/ml antibody; the fixative is unreported (catalog IHC caption). For IF/ICC, choose A01319-1 because both applications are listed and its IF image shows U20S cells (catalog applications; catalog IF caption). For cross-species work, A01319-1 lists human, mouse, and rat reactivity, while the supplied IHC image demonstrates human tissue only (catalog reactivity; catalog IHC caption).

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

References

UniProt Consortium. UniProt entry P30291 (WEE1_HUMAN, Wee1-like protein kinase).
Human Protein Atlas. WEE1 tissue IHC expression (reliability: Approved).
Human Protein Atlas. WEE1 subcellular location (ICC-IF): Localized to the nucleoli..
Human Protein Atlas. WEE1 antibody validation summary (2 antibodies).
Integrated functional, gene expression and genomic analysis for the identification of cancer targets. PloS one 2009 — PMC2663812.
High expression of wee1 is associated with malignancy in vulvar squamous cell carcinoma patients. BMC cancer 2013 — PMC3751528.
Combined evaluation of both WEE1 and phosphorylated cyclin dependent kinase 1 expressions in oral squamous cell carcinomas predicts cancer recurrence and progression. Journal of dental sciences 2022 — PMC9588842.
Targeting WEE1 to enhance conventional therapies for acute lymphoblastic leukemia. Journal of hematology & oncology 2018 — PMC6090987.
PubMed PMID:7743995 — UniProt-cited evidence.
PubMed PMID:11528126 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.