AK1 / Adenylate kinase isoenzyme 1 · IHC design guide

Design Immunohistochemistry for AK1

Plan AK1 IHC in paraffin sections using the expected cytoplasmic staining in skeletal myocytes and ciliated cells (HPA tissue IHC). This guide covers the catalog antibody’s chromogenic IHC workflow (datasheet PB10033).

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

Immunohistochemistry protocol sheet for AK1 (IHC for AK1): expected localisation Cytoplasmic staining in variable cell types (HPA tissue IHC), antibody PB10033, validated IHC image, and IHC protocol steps — Printable **AK1** IHC protocol sheet — expected localisation Cytoplasmic staining in variable cell types (HPA tissue IHC), antibody PB10033, controls and protocol steps. Open the full AK1 IHC guide →

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AK1 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 variable cell types (HPA tissue IHC)
	Staining pattern	Cytoplasmic staining in myocytes and ciliated cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB10033)
	Positive control ⓘ	Choroid plexus+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	Cell-type composition can change apparent staining (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	No annotated isoforms; one 1–194 chain (UniProt)

Section 1

Recommended AK1 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet PB10033) with a published AK1 protocol for clinical breast cancer lesions (PMC2882958).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human colon cancer tissue; fixative not specified (datasheet PB10033)
Fixation	Image fixative and duration unreported (datasheet PB10033); 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 PB10033); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB10033)
Primary antibody	Rabbit anti-AK1, 2-5μg/ml (datasheet PB10033)
Primary incubation	Overnight at 4 °C (datasheet PB10033)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet PB10033)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	AK1-positive staining in ependymal cells of choroid plexus (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression at variable levels in several different cell types, most abundant in skeletal muscle, fallopian tube, choroid plexus and efferent ducts. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval (datasheet PB10033). For optimization, compare the published retrieval solution at 95°C for 40 min (PMC2882958).

Section 2

What Is the Expected AK1 Staining Pattern?

AK1 is cytoplasmic and has no transmembrane segment (UniProt P00568). In paraffin tissue sections, expect variable cytoplasmic staining, with high staining reported in skeletal myocytes, choroid plexus ependymal cells, and ciliated cells of the fallopian tube and efferent ducts (HPA: tissue IHC). HPA rates the tissue profile Enhanced, while noting medium consistency between antibody staining and RNA expression (HPA: reliability).

What am I looking at on my slide?
Strong cytoplasmic staining in skeletal myocytes or choroid plexus ependymal cells.	This matches cells scored High by HPA and the cytoplasmic location assigned by UniProt (HPA: tissue IHC; UniProt P00568). Judge the distribution within the expected cells, not intensity alone (general IHC practice).
Strong, predominantly nuclear staining in otherwise positive cells.	A predominantly nuclear pattern conflicts with the expected cytoplasmic location (UniProt P00568; HPA: tissue IHC). Check whether staining persists with a second validated antibody and appropriate detection controls before calling it AK1 (general IHC practice).
Prominent staining of adipocytes or appendix glandular cells.	HPA reports AK1 as Not detected in those cell types (HPA: tissue IHC). Unexpected signal raises concern for antibody cross-reactivity or endogenous detection activity; compare a no-primary control and expected positive tissue (general IHC practice).
Diffuse colour across tissue and blank areas, with little cell definition.	This cannot establish the reported cell-specific cytoplasmic pattern (HPA: tissue IHC). Review blocking, washes, detection reagent and counterstain using controls processed alongside the section (general IHC practice).
No staining in skeletal myocytes or choroid plexus ependymal cells.	Both are scored High by HPA, so a blank result warrants an assay check (HPA: tissue IHC). Confirm tissue identity and control performance, then review antibody dilution, retrieval and detection conditions as general IHC variables (general IHC practice).

💡Expected AK1 appearanceCall an IHC result positive when expected cells show defined cytoplasmic staining, often strong in skeletal myocytes or choroid plexus ependymal cells; isolated nuclear or cell-inappropriate colour needs control checks (HPA: tissue IHC; UniProt P00568; general IHC practice).

How each factor affects the staining
Tissue and cell choice	HPA scores skeletal myocytes, choroid plexus ependymal cells, and fallopian tube ciliated cell bodies High, but thyroid glandular cells Medium and bronchial ciliated cell bodies Low (HPA: tissue IHC). Set expectations by cell type rather than by organ alone.
Subcellular distribution	AK1 is cytoplasmic and lacks a transmembrane segment (UniProt P00568). HPA also reports High staining in nasopharyngeal cilia axonemes, so that specific reported pattern should not be dismissed solely because most tissue staining is cytoplasmic (HPA: tissue IHC).
Antibody validation	HPA marks IHC staining with HPA006456 and CAB009893 as Enhanced (HPA: antibodies). Its tissue profile still has medium consistency with RNA expression; use the reported pattern as an expectation, not proof that every positive pixel is AK1 (HPA: reliability).
What should IF/ICC show?	Mainly cytosolic signal, with an additional annulus location reported by HPA (HPA: subcellular ICC-IF). This is an IF/ICC localisation reference; score paraffin IHC against the tissue cell patterns above (HPA: tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known-positive tissue is blank.	The result conflicts with cells scored High by HPA, but the image alone does not identify which assay step failed (HPA: tissue IHC).	Check the positive control and tissue identity; then review primary dilution, retrieval and chromogenic detection settings as general IHC variables (general IHC practice).
Staining is mainly nuclear.	The compartment disagrees with UniProt cytoplasm and HPA's mainly cytosolic ICC-IF localisation (UniProt P00568; HPA: subcellular ICC-IF).	Inspect the no-primary control and compare with a second IHC-validated antibody where available (HPA: antibodies; general IHC practice).
Unexpected cells stain strongly.	Adipocytes and appendix glandular cells are scored Not detected, making strong signal there questionable (HPA: tissue IHC).	Check no-primary and expected-positive controls for endogenous detection activity or nonspecific staining; compare cell distribution before scoring (general IHC practice).
Most of the section has diffuse colour.	Widespread, poorly defined signal obscures the variable cell-specific cytoplasmic pattern reported by HPA (HPA: tissue IHC).	Review reagent blocking, wash steps, detection background and counterstain against a matched control section (general IHC practice).
Ciliated epithelium looks inconsistent across sites.	HPA scores fallopian tube ciliated cell bodies High, bronchial ciliated cell bodies Low, and nasopharyngeal cilia axonemes High (HPA: tissue IHC).	Record the organ and whether colour is in the cell body or axoneme; compare only the corresponding HPA pattern (HPA: tissue IHC).
IF/ICC signal appears outside the cytosol.	HPA reports an additional annulus location, but its main ICC-IF location is cytosol (HPA: subcellular ICC-IF).	Check whether the signal matches the reported annulus pattern and assess imaging controls; use the separate IF/ICC guide for assay setup (HPA: subcellular ICC-IF; general IF practice).

Source: UniProt P00568 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AK1 staining

IHC analysis of AK1 using anti-AK1 antibody (PB10033). AK1 was detected in a paraffin-embedded section of human colon cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AK1 Antibody (PB10033) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

PB10033 · IHCIHC analysis of AK1 using anti-AK1 antibody (PB10033). AK1 was detected in a paraffin-embedded section of human colon cancer tissue. Heat me

IHC analysis of AK1 using anti-AK1 antibody (PB10033). AK1 was detected in a paraffin-embedded section of human lung adenocarcinoma tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH 8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2 μg/ml rabbit anti-AK1 Antibody (PB10033) overnight at 4°C. Peroxidase Conjugated Goat Anti-rabbit IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Rabbit IgG Super Vision Assay Kit (Catalog # SV0002) with DAB as the chromogen.

PB10033 · IHCIHC analysis of AK1 using anti-AK1 antibody (PB10033). AK1 was detected in a paraffin-embedded section of human lung adenocarcinoma tissue.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000106992-AK1/tissue/cerebral+cortex#imid_2810936

HPA · Choroid plexusHuman Protein Atlas IHC, Choroid plexus. Source: https://www.proteinatlas.org/ENSG00000106992-AK1/tissue/choroid+plexus#imid_4335

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000106992-AK1/tissue/colon#imid_2810854

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000106992-AK1/tissue/liver#imid_2810871

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006456; green = AK1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for AK1 IHC & IF

🧪Run fallopian tube first; ciliated cell bodies should stain (HPA: High in fallopian tube ciliated cells). Use adipose tissue as the negative tissue; adipocytes are listed as not detected (HPA: Not detected in adipocytes). On the positive slide, record unstained neighboring cells as internal background comparators without assigning them a negative cell identity from the supplied HPA rows.

Positive control tissue: Choroid plexus (Ependymal cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AK1 in A-431, U-251MG, U2OS, ASC52telo, Sperm, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a matched nonimmune rabbit IgG isotype control, since the catalog antibody is rabbit-derived (PB10033 tissue-IHC caption). Include AK1-knockout tissue as a biological negative if available, and quench endogenous peroxidase before HRP/DAB detection (PB10033 tissue-IHC caption: HRP/DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact PB10033 tissue-IHC caption does not state the fixative. That paraffin-section example used heat retrieval in EDTA at pH 8.0, but it does not establish whether retrieval is essential; the supplied evidence also does not establish that frozen sections or IF are easier (PB10033 tissue-IHC caption). In fallopian tube, distinguish staining in ciliated cell bodies from deposits on the luminal surface when scoring (HPA: High in ciliated cells, cell body).

HPA tissue IHC evidence for AK1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — Medium consistency between antibody staining and RNA expression data. Protein expression in respiratory epithelia can be correlated with lung RNA-seq 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
Choroid plexus	Ependymal cells	High	Protein (IHC)	HPA →
Efferent ducts	Ciliated cells	High	Protein (IHC)	HPA →
Fallopian tube	Ciliated cells (cell body)	High	Protein (IHC)	HPA →
Lung	Alveolar cells type II	High	Protein (IHC)	HPA →
Nasopharynx	Ciliated cells (cilia axoneme)	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Appendix	Glandular cells	Not detected	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →

Section 3

Advanced AK1 IHC Tips

Troubleshoot AK1 staining in paraffin sections by checking retrieval, cytoplasmic localisation, cell type and detection controls before comparing staining intensity.

How should I adjust retrieval when AK1 staining is weak in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 for AK1 paraffin sections (datasheet PB10033). The documented example then used 10% goat serum, 2 μg/ml primary antibody overnight at 4°C, and a peroxidase-based DAB readout (datasheet PB10033). If staining remains weak, check whether heating and cooling were consistent across slides, then adjust retrieval duration in a small side-by-side series while keeping antibody and detection conditions fixed (standard IHC practice). Judge recovery by cytoplasmic signal in expected cells and by preserved morphology, because diffuse tissue staining alone does not establish AK1 specificity (UniProt P00568 subcellular; HPA tissue IHC; standard IHC practice).

Can fixation explain weak or uneven AK1 IHC staining?

AK1-specific fixation sensitivity is unknown: the selected paraffin-section caption does not state its fixative (datasheet PB10033). Record each specimen’s fixative and fixation duration, then compare similarly processed sections before attributing differences to AK1 abundance (standard IHC practice). Keep the documented EDTA retrieval at pH 8.0 and the 2 μg/ml primary-antibody condition constant during that comparison (datasheet PB10033). If morphology is poor or staining varies within a block, inspect section quality and processing records, and include a consistently processed reference section in the same run (standard IHC practice). The HPA tissue pattern provides expression context, not evidence of fixation sensitivity (HPA tissue IHC).

Which staining compartment should count as AK1 positive?

Score predominantly cytoplasmic staining in intact cells, consistent with AK1’s cytoplasmic annotation and the HPA cytosol localisation (UniProt P00568 subcellular; HPA subcellular). In tissue, skeletal-muscle myocytes and fallopian-tube ciliated-cell bodies are reported as high-expression examples for comparison (HPA tissue IHC). Distinguish cell-body staining from staining confined to a luminal border, pigment or debris by reviewing morphology alongside the counterstain (standard IHC practice). HPA also reports AK1 in nasopharyngeal cilia axonemes and an additional annulus location in subcellular imaging, so assess those specialised structures in their own anatomical context (HPA tissue IHC; HPA subcellular). Record compartment and cell type separately from intensity (standard IHC practice).

How should I investigate an unexpected AK1 staining pattern?

The supplied AK1 record lists one 194-aa chain, no annotated isoforms, no transmembrane segment and no signal peptide (UniProt P00568). Thus, an apparent membrane-only pattern warrants scrutiny of morphology, detection controls and antibody specificity before assigning a new AK1 localisation (UniProt P00568 topology; standard IHC practice). The record lists N-terminal acetylation at residue 1 and phosphoserine at residue 38, but supplies no antibody epitope or evidence that either modification changes staining (UniProt P00568 modified residues; datasheet PB10033). Check the antibody’s documented immunogen or epitope if available, and compare cytoplasmic staining with expected positive and low-expression cells on the same run (standard IHC practice; HPA tissue IHC).

How can I use IF to check a puzzling AK1 IHC pattern?

Use IF as an orthogonal localisation check, while keeping the paraffin-section chromogenic result as the IHC readout (standard IF/IHC practice). Multiplex AK1 with a marker for the expected cell type, such as a validated ciliated-cell marker when examining fallopian-tube ciliated cells; HPA reports high AK1 staining in those cell bodies (HPA tissue IHC; standard IF practice). Choose spectrally separated fluorophores after inspecting unstained tissue for autofluorescence, and include single-stain controls for bleed-through (standard IF practice). AK1 is cytosolic and has no transmembrane segment, so permeabilise fixed cells sufficiently to expose an intracellular epitope and verify that the treatment preserves cell morphology (UniProt P00568 subcellular/topology; standard IF practice).

What should I check when DAB background obscures AK1 staining?

Inspect no-primary and secondary-only controls to determine whether diffuse DAB signal persists without the AK1 antibody (standard IHC practice). Block endogenous peroxidase before HRP detection, and check wash stringency, secondary-antibody binding and DAB development time when background is widespread (standard IHC practice). The documented paraffin example used 10% goat serum, a peroxidase-conjugated secondary for 30 minutes at 37°C, and DAB; these are documented conditions, not proof that background will be absent in other specimens (datasheet PB10033). Compare background with expected cytoplasmic staining in intact cells, and avoid interpreting edge deposits, folds or necrotic material as AK1-positive cells (UniProt P00568 subcellular; standard IHC practice).

How should I quantify AK1 staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and cytoplasmic compartment before scoring, because AK1 is cytoplasmic and HPA reports variable expression across cell types (UniProt P00568 subcellular; HPA tissue IHC). For cellular staining, report the percentage of positive cells and an H-score from 0–300, calculated from the percentages at intensity scores 0–3 (standard IHC practice). If counting discrete positive cells, report density per mm² of viable, evaluable tissue and specify the analysed region (standard IHC practice). Normalise comparisons to the same cell type or tissue area, section processing, staining run and image-acquisition settings, while excluding folds and necrosis by a prespecified rule (standard IHC practice).

How can I distinguish true AK1 staining from artefact?

A credible AK1 result follows cell anatomy and shows cytoplasmic signal in intact cells, consistent with the UniProt location and HPA tissue profile (UniProt P00568 subcellular; HPA tissue IHC). High staining in skeletal-muscle myocytes or choroid-plexus ependymal cells offers an expression reference, whereas adipocytes in adipose tissue are reported as not detected (HPA tissue IHC). Investigate signal restricted to section edges, necrotic regions or a discordant compartment using morphology and no-primary controls; endogenous peroxidase can also produce DAB signal if inadequately blocked (standard IHC practice). Interpret unexpected cells cautiously because the HPA tissue assessment has Enhanced reliability but reports only medium consistency between staining and RNA data (HPA tissue IHC).

Boster reagents

Best AK1 / Adenylate kinase isoenzyme 1 IHC Antibodies

PB10033 has IHC images from paraffin sections of human colon cancer and lung adenocarcinoma, and mouse and rat heart; no IF images are supplied (catalog IHC/IF captions).

Real IHC data

Anti-Adenylate Kinase 1/AK1 Antibody ®

Cat # PB10033

View product →

PB10033 is listed for IHC in human, mouse and rat (catalog applications/reactivity). Its IHC captions show paraffin sections of human colon cancer, human lung adenocarcinoma, mouse heart and rat heart (PB10033 IHC captions).

Which to pick: Choose PB10033 for paraffin-section IHC; its own captions document these sections with EDTA retrieval and 2 μg/ml primary antibody (PB10033 IHC captions). For cross-species IHC, PB10033 lists human, mouse and rat reactivity (catalog reactivity); the captions do not report a fixative (PB10033 IHC captions). No IF/ICC application or image is supplied for PB10033, so an IF/ICC choice is unsupported by this catalog (catalog applications/IF captions).

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

References

UniProt Consortium. UniProt entry P00568 (KAD1_HUMAN, Adenylate kinase isoenzyme 1).
Human Protein Atlas. AK1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. AK1 subcellular location (ICC-IF): Mainly localized to the cytosol. In addition localized to the annulus..
Human Protein Atlas. AK1 antibody validation summary (2 antibodies).
Genome-wide analysis of mRNA and microRNA expression in colorectal cancer and adjacent normal mucosa. The journal of pathology. Clinical research 2022 — PMC9161315.
Proteome-wide profiling of the MCF10AT breast cancer progression model. PloS one 2010 — PMC2882958.
Inhibition of Sirtuin 2 with Sulfobenzoic Acid Derivative AK1 is Non-Toxic and Potentially Neuroprotective in a Mouse Model of Frontotemporal Dementia. Frontiers in pharmacology 2012 — PMC3298895.
4EBP1/eIF4E and p70S6K/RPS6 axes play critical and distinct roles in hepatocarcinogenesis driven by AKT and N-Ras proto-oncogenes in mice. Hepatology (Baltimore, Md.) 2015 — PMC4280310.
PubMed PMID:183954 — UniProt-cited evidence.
PubMed PMID:2542324 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.