PKLR / Pyruvate kinase PKLR · IHC design guide

Design Immunohistochemistry for PKLR

Plan chromogenic PKLR IHC-P around strong cytoplasmic staining in hepatocytes and proximal renal tubules (HPA tissue IHC). Use hematopoietic cells as a medium-staining reference, and consider presumed off-target staining when scoring (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for PKLR (IHC for PKLR): expected localisation Cytoplasmic in hepatocytes and renal tubule cells (HPA tissue IHC), antibody PB9499, validated IHC image, and IHC protocol steps — Printable **PKLR** IHC protocol sheet — expected localisation Cytoplasmic in hepatocytes and renal tubule cells (HPA tissue IHC), antibody PB9499, controls and protocol steps. Open the full PKLR IHC guide →

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PKLR 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 in hepatocytes and renal tubule cells (HPA tissue IHC)
	Staining pattern	Cytoplasmic hepatocytes, renal tubules and hematopoietic cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB9499)
	Positive control ⓘ	Kidney+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	Presumed off-target staining may confound scoring (HPA tissue IHC)
	Regulation	RNA enhanced in marrow, kidney and liver (HPA tissue RNA)
	Isoform / epitope	2 R/L isoforms; antibody epitope coverage is unknown (UniProt)

Section 1

Recommended PKLR IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet: PB9499) with two published PKLR staining protocols (PMC8636031; PMC6603390).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet PB9499)
Fixation	Image fixative and duration unreported (datasheet PB9499); 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 PB9499); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9499)
Primary antibody	Rabbit anti-PKLR, 0.5-1μg/ml (datasheet PB9499)
Primary incubation	Overnight at 4 °C (datasheet PB9499)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9499)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	PKLR-positive staining in proximal tubules (cell body) of kidney (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression mainly in hepatocytes, hematopoietic cells, intestines and cells in renal tubules. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 for the catalog antibody (datasheet: PB9499). The tissue microarray protocol used citrate at pH 6 (PMC6603390).

Section 2

What Is the Expected PKLR Staining Pattern?

PKLR should appear predominantly in the cytoplasm of hepatocytes and renal proximal tubule cells, with staining also reported in marrow hematopoietic cells and intestinal endocrine cells (HPA: tissue IHC). HPA rates the tissue pattern Enhanced, while noting presumed off-target binding that was disregarded (HPA: tissue IHC). PKLR has no annotated transmembrane segment; UniProt does not assign a subcellular location (UniProt P30613 topology and subcellular record).

What am I looking at on my slide?
Strong cytoplasmic stain in hepatocytes or renal proximal tubule cell bodies.	This matches HPA High staining in both cell populations (HPA: liver and kidney IHC). Compare cellular distribution with an appropriate negative control before calling a specimen positive (standard IHC practice).
Predominantly nuclear, membranous, or extracellular stain.	These compartments do not match the reported cytoplasmic tissue pattern or approved cytosolic ICC-IF location (HPA: tissue IHC and subcellular). Check whether the signal persists in a negative control; a mismatched compartment alone does not identify its cause (standard IHC practice).
Strong stain in cells expected to be negative, such as adipocytes or adrenal glandular cells.	HPA reports PKLR as Not detected in those cells (HPA: adipose tissue and adrenal gland IHC). Consider cross-reactivity or endogenous detection activity, particularly if control slides show the same signal (standard IHC practice); HPA also notes presumed off-target binding in its tissue assessment (HPA: tissue IHC).
Diffuse colour across tissue and blank areas, obscuring cell boundaries.	This is difficult to score as cell-specific PKLR staining (standard IHC practice). Inspect the no-primary control for detection-system background and review blocking and washing; the HPA expression profile does not explain diffuse slide-wide colour (standard IHC practice; HPA: tissue IHC).
No stain in hepatocytes or renal proximal tubules on the same run.	Both are HPA High reference populations (HPA: liver and kidney IHC). Check the positive-control section, antibody dilution, retrieval, and detection steps before interpreting an unstained test section; the supplied sources give no PKLR-specific fixation-sensitivity finding (standard IHC practice; UniProt P30613 and HPA: tissue IHC).

💡Expected PKLR appearanceCall a convincing positive when hepatocytes or renal proximal tubule cell bodies show strong cytoplasmic stain (HPA: High, liver and kidney IHC); dominant nuclear, membranous, or negative-control stain is a warning pattern (HPA: tissue IHC and subcellular; standard IHC practice).

How each factor affects the staining
Cell compartment	HPA describes cytoplasmic tissue expression and approves Cytosol in ICC-IF (HPA: tissue IHC and subcellular). UniProt provides no subcellular annotation and lists no transmembrane segment, so the HPA observations anchor the localisation call (UniProt P30613 topology and subcellular record).
Cell population and intensity	HPA scores hepatocytes and renal proximal tubules High; marrow hematopoietic and duodenal or small-intestinal endocrine cells Medium (HPA: tissue IHC). Colon endocrine cells are Low, so a weak result there has less diagnostic weight than a High reference population (HPA: tissue IHC).
Antibody validation	The tissue profile is rated Enhanced, with presumed off-target binding observed and disregarded (HPA: tissue IHC). HPA lists Enhanced IHC for HPA006653, CAB034376, and CAB034378; agreement supports the reported pattern but does not prove every stain in a new specimen is specific (HPA: antibody validation).
Isoforms and processing	UniProt lists R-type and L-type isoforms and one annotated chain spanning residues 1–574, with no signal peptide or propeptide (UniProt P30613 isoforms and processing). These entries do not establish isoform-specific IHC staining or an antibody's epitope; interpret the observed cells against HPA tissue controls (HPA: tissue IHC).
IF/ICC Q&A: What localisation should a separate IF/ICC guide assess?	Cytosol is the approved location; HPA lists A-431, Hep-G2, and K-562 ICC-IF image sets, and HPA006653 has Approved ICC status (HPA: subcellular and antibody validation). Those data support a localisation check, not an IF/ICC protocol option on this IHC page.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
High reference tissue is blank.	A run-level staining failure is possible when HPA High hepatocytes or proximal tubules are unstained (HPA: tissue IHC).	Review the positive control, catalog antibody's IHC-P dilution and retrieval directions, and detection reagents (standard IHC practice); do not infer PKLR-specific fixation sensitivity from these sources.
Only nuclei are strongly coloured.	The compartment conflicts with cytoplasmic tissue staining and approved cytosolic ICC-IF localisation (HPA: tissue IHC and subcellular).	Compare no-primary and known-positive sections, then reassess scoring and staining specificity (standard IHC practice).
Adipocytes stain strongly.	HPA records adipocytes as Not detected; off-target or detection-system signal is possible (HPA: adipose tissue IHC; standard IHC practice).	Check a no-primary control and compare the suspect area with a High reference tissue (standard IHC practice; HPA: liver or kidney IHC).
Colour appears without primary antibody.	A no-primary signal indicates background from the detection workflow, such as endogenous activity or nonspecific reagent deposition (standard IHC practice).	Review the relevant endogenous-activity block, reagent controls, and washing for the chosen chromogen system (standard IHC practice).
All cells show weak, diffuse colour.	The result lacks the HPA cell-selective pattern and may reflect background rather than interpretable PKLR signal (HPA: tissue IHC; standard IHC practice).	Review no-primary controls, blocking, washes, and the catalog antibody's IHC-P dilution (standard IHC practice).
Intestinal endocrine cells look weaker than hepatocytes.	HPA scores duodenal and small-intestinal endocrine cells Medium, versus High hepatocytes (HPA: tissue IHC).	Score by cell type and compare with the expected level; investigate only if positive controls also fail (HPA: tissue IHC; standard IHC practice).

Source: UniProt P30613 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing PKLR staining

IHC analysis of PKLR using anti-PKLR antibody (PB9499). PKLR was detected in a paraffin-embedded section of human intestinal 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 1 μg/ml rabbit anti-PKLR Antibody (PB9499) 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.

PB9499 · IHCIHC analysis of PKLR using anti-PKLR antibody (PB9499). PKLR was detected in a paraffin-embedded section of human intestinal cancer tissue.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000143627-PKLR/tissue/cerebral+cortex#imid_14407044

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000143627-PKLR/tissue/colon#imid_14406954

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000143627-PKLR/tissue/liver#imid_14406955

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000143627-PKLR/tissue/kidney#imid_14406972

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA006653; green = PKLR, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for PKLR IHC & IF

🧪Run liver first: hepatocytes should stain (HPA: High in hepatocytes). Use adipose tissue as the negative tissue, with adipocytes expected to be unstained (HPA: Not detected in adipocytes); on the liver slide, compare hepatocytes with cells outside the hepatocyte compartment as an internal background reference, without assuming those cells are verified PKLR-negative.

Positive control tissue: Kidney (Proximal tubules (cell body), HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show PKLR in A-431, Hep-G2, K-562, with annotated localisation: Cytosol (approved) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control, a matched rabbit isotype control, and PKLR-knockout material as a biological negative (caption: rabbit anti-PKLR antibody; standard IHC practice). For liver, control endogenous peroxidase and biotin signal when using the caption’s biotin–streptavidin and DAB detection (caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected PB9499 paraffin-section caption does not state a fixative (caption: fixative unreported). The demonstrated IHC procedure uses heat retrieval in EDTA at pH 8.0, but the evidence does not establish whether retrieval is required or whether frozen sections or IF/ICC are easier (caption: EDTA heat retrieval; HPA: cytosolic ICC-IF localization). In liver, endogenous peroxidase or biotin can complicate chromogenic interpretation (standard IHC practice).

HPA tissue IHC evidence for PKLR

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High consistency between antibody staining and RNA expression data. Presumed off target binding observed and disregarded.). 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
Kidney	Proximal tubules (cell body)	High	Protein (IHC)	HPA →
Liver	Hepatocytes	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	Medium	Protein (IHC)	HPA →
Duodenum	Endocrine cells	Medium	Protein (IHC)	HPA →
Small intestine	Endocrine cells	Medium	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 →
Appendix	Endocrine cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced PKLR IHC Tips

Troubleshoot PKLR chromogenic IHC in paraffin sections using the catalog antibody’s reported conditions and tissue staining as reference points.

Which retrieval condition should I try first for weak PKLR staining?

Start with heat-mediated antigen retrieval in EDTA at pH 8.0 (datasheet PB9499). The reported paraffin-section example used this retrieval before overnight incubation at 4°C with 1 μg/ml catalog antibody (caption PB9499). If staining remains weak, compare retrieval duration on serial sections while holding antibody concentration, detection and development constant (standard IHC practice). Include liver hepatocytes or kidney proximal tubules as an expected positive reference, and examine both staining intensity and tissue preservation (HPA: High in hepatocytes and proximal tubules; standard IHC practice). Do not interpret stronger staining alone as improved specificity; compare its cell distribution with the reference section and a no-primary control (standard IHC practice).

Can fixation explain variable PKLR staining across paraffin blocks?

PKLR-specific sensitivity to fixative type or fixation duration is unknown from the supplied evidence; the selected paraffin-section caption does not state its fixative (caption PB9499). Record each block’s fixative and processing history before attributing a staining difference to antigen abundance (standard IHC practice). Compare serial sections under the same EDTA pH 8.0 retrieval, 1 μg/ml primary concentration and overnight 4°C incubation used in the reported example (caption PB9499). If a block stains poorly, assess morphology and an independently validated control stain alongside PKLR to check general tissue quality (standard IHC practice). Neither cytosolic staining nor annotated phosphorylation sites establish a PKLR-specific fixation effect (HPA: Cytosol; UniProt P30613 modified residues).

Where should convincing PKLR signal appear in a tissue section?

Expect predominantly cytoplasmic staining in relevant cells, with cytosol reported as the approved subcellular location (HPA: tissue IHC profile; HPA: Cytosol). High staining in hepatocytes and kidney proximal tubules provides useful cell-level reference patterns (HPA: High in hepatocytes and proximal tubules). PKLR has no annotated transmembrane segment or signal peptide, so a sharp membrane-only or extracellular pattern warrants investigation rather than automatic acceptance (UniProt P30613 topology and processing). Compare suspicious areas with adjacent intact tissue, a no-primary control and the expected cytoplasmic pattern before scoring (standard IHC practice; HPA: Cytosol). Keep cell identity and compartment separate in your notes: a positive-looking area can contain cells outside the expected reference populations (HPA: tissue IHC profile).

Can this stain distinguish PKLR’s L-type and R-type isoforms?

Do not assign an isoform from staining alone: PKLR has 2 annotated isoforms, L-type and R-type, while the supplied PB9499 caption does not identify an isoform-discriminating epitope (UniProt P30613 isoforms; caption PB9499). Before making an isoform claim, obtain the antibody’s immunogen or epitope sequence and compare it with both isoform sequences (standard antibody validation practice). The record also lists phosphorylated residues, but it does not establish phosphorylation-dependent recognition by this antibody (UniProt P30613 modified residues; caption PB9499). If isoform assignment matters, validate it with an independent isoform-specific assay on matched material and report the IHC result as PKLR staining until that comparison is complete (standard antibody validation practice).

How should I adapt PKLR localisation checks for multiplex immunofluorescence?

On this IHC page, use IF as a separate localisation check: an approved cytosolic PKLR location and ICC/IF images in A-431, Hep-G2 and K-562 are reported (HPA: subcellular). For tissue multiplexing, pair PKLR with a marker that identifies the expected cell population, such as a validated hepatocyte or proximal-tubule marker, and assess cellular overlap rather than total field brightness (HPA: High in hepatocytes and proximal tubules; standard IF practice). Choose fluorophores after examining tissue autofluorescence in unstained controls, and separate channels sufficiently to assess bleed-through (standard IF practice). Use permeabilisation appropriate for an intracellular cytosolic epitope, then check morphology and signal with and without the primary antibody (HPA: Cytosol; standard IF practice).

What should I check when PKLR DAB staining is diffuse?

First compare the section with a no-primary control and inspect whether diffuse colour follows tissue edges, damaged regions or all cell types (standard IHC practice). The reported PB9499 example used 10% goat serum blocking, biotinylated secondary antibody, a streptavidin-biotin complex and DAB development (caption PB9499). For that detection format, check peroxidase blocking and consider endogenous biotin as possible background sources using appropriate control sections (standard IHC practice). Reassess primary concentration and DAB development against the reported 1 μg/ml starting condition, changing one variable at a time (caption PB9499; standard IHC practice). Retain staining only when the cellular pattern remains interpretable relative to cytoplasmic positive-reference cells (HPA: tissue IHC profile; standard IHC practice).

How should I score PKLR staining across samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population before scoring and record cytoplasmic intensity separately from the percentage of positive cells (HPA: cytoplasmic tissue IHC profile; standard IHC scoring practice). An H-score can combine those measures as intensity from 0–3 multiplied by the percentage at each intensity, yielding 0–300 (standard IHC scoring practice). Alternatively, report percentage-positive cells or positive-cell density per mm², with a fixed positivity threshold and sampled area across sections (standard IHC scoring practice). Normalise counts to the number of evaluable cells or evaluable tissue area within the same defined compartment, excluding necrosis and folds (standard IHC scoring practice). Use hepatocytes or proximal tubules as reference populations when present, and document their scores independently from the study population (HPA: High in hepatocytes and proximal tubules).

How can I separate true PKLR staining from artefact?

Give greatest weight to cytoplasmic staining in identifiable expected cells, especially hepatocytes or kidney proximal tubules (HPA: cytoplasmic expression; HPA: High in hepatocytes and proximal tubules). Treat a membrane-only pattern, staining confined to section edges or necrotic areas, and signal in the no-primary control as reasons to investigate artefact (UniProt P30613 topology; standard IHC practice). Check peroxidase blocking when DAB colour appears independent of primary antibody, particularly in enzyme-rich or damaged regions (standard IHC practice). HPA reports enhanced tissue-IHC reliability while also noting presumed off-target binding that was disregarded, so its pattern is a reference rather than proof for every cell (HPA: reliability description). Confirm surprising cell-level findings with controls or independent evidence before assigning biological meaning (standard IHC practice).

Boster reagents

Best PKLR / Pyruvate kinase PKLR IHC Antibodies

PB9499 has a real IHC image from a human intestinal cancer paraffin section (catalog: IHC caption). Its listed reactivity is Human, Mouse and Rat (catalog: reactivity).

Real IHC data

Anti-PKLR Antibody ®

Cat # PB9499

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PB9499 will render with an IHC figure showing staining in a human intestinal cancer paraffin section (catalog: IHC caption). Its listed applications are IHC and WB, while the figure documents IHC in human tissue only (catalog: applications; IHC caption).

Which to pick: Choose PB9499 for paraffin-section IHC; its own caption documents EDTA retrieval at pH 8.0 and incubation at 1 μg/ml overnight at 4°C (catalog: PB9499 IHC caption). No IF/ICC option is documented (catalog: PB9499 applications; IF images). PB9499 lists Human, Mouse and Rat reactivity, but its IHC figure shows human tissue only; the fixative is unreported (catalog: PB9499 reactivity; IHC caption).

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

References

UniProt Consortium. UniProt entry P30613 (KPYR_HUMAN, Pyruvate kinase PKLR).
Human Protein Atlas. PKLR tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. PKLR subcellular location (ICC-IF): Localized to the cytosol..
Human Protein Atlas. PKLR antibody validation summary (3 antibodies).
Combined Analysis of Expression Profiles in a Mouse Model and Patients Identified BHMT2 as a New Regulator of Lipid Metabolism in Metabolic-Associated Fatty Liver Disease. Frontiers in cell and developmental biology 2021 — PMC8636031.
Tumoral Pyruvate Kinase L/R as a Predictive Marker for the Treatment of Renal Cancer Patients with Sunitinib and Sorafenib. Journal of Cancer 2019 — PMC6603390.
Differential responses on energy metabolic pathway reprogramming between genotoxic and non-genotoxic hepatocarcinogens in rat liver cells. Journal of toxicologic pathology 2019 — PMC6831489.
Downregulation of miR-122-5p Activates Glycolysis via PKM2 in Kupffer Cells of Rat and Mouse Models of Non-Alcoholic Steatohepatitis. International journal of molecular sciences 2022 — PMC9101520.
PubMed PMID:1896471 — UniProt-cited evidence.
PubMed PMID:3126495 — UniProt-cited evidence.
PubMed PMID:1445295 — UniProt-cited evidence.