AGPS / Alkyldihydroxyacetonephosphate synthase, peroxisomal · IHC design guide

Design Immunohistochemistry for AGPS

Plan AGPS staining in paraffin sections with the IHC-validated antibody at 2–5 μg/ml (datasheet: IHC-P). Use the granular cytoplasmic pattern and strong ciliary-rootlet staining to interpret tissue sections (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for AGPS (IHC for AGPS): expected localisation Granular cytoplasm in tissue (HPA tissue IHC), antibody A03481-1, validated IHC image, and IHC protocol steps — Printable **AGPS** IHC protocol sheet — expected localisation Granular cytoplasm in tissue (HPA tissue IHC), antibody A03481-1, controls and protocol steps. Open the full AGPS IHC guide →

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AGPS 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	Granular cytoplasm in tissue (HPA tissue IHC)
	Staining pattern	Granular cytoplasm in glandular cells; ciliary rootlets can stain strongly (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A03481-1)
	Positive control ⓘ	Bronchus+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	Strong ciliary-rootlet staining can alter the expected pattern (HPA tissue IHC)
	Regulation	Baseline abundance varies by cell type (HPA tissue IHC)
	Isoform / epitope	No annotated isoforms; mature chain starts at 59, relevant to N-terminal epitopes (UniProt)

Section 1

Recommended AGPS IHC & IF Protocols

The catalog antibody protocol uses EDTA pH 8.0 heat retrieval (datasheet A03481-1). The published protocols below cover renal biopsies (PMC12460175) and prostate tissues (PMC10782585).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human adenocarcinoma of lung tissue; fixative not specified (datasheet A03481-1)
Fixation	Image fixative and duration unreported (datasheet A03481-1); 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 A03481-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A03481-1)
Primary antibody	Rabbit anti-AGPS, 2-5 μg/ml (datasheet A03481-1)
Primary incubation	Overnight at 4 °C (datasheet A03481-1)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A03481-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	AGPS-positive staining in ciliated cells (ciliary rootlets) of bronchus (HPA tissue IHC: High). HPA tissue profile: Ubiquitous granular cytoplasmic expression, most abundant in glandular cells. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet A03481-1); neither published excerpt specifies a retrieval method (PMC12460175; PMC10782585).

Section 2

What Is the Expected AGPS Staining Pattern?

AGPS is annotated in peroxisomes and at the peroxisome membrane, with no transmembrane segment (UniProt O00116). In paraffin-section IHC, expect granular cytoplasmic staining, most abundant in glandular cells; ciliary rootlets in bronchus and fallopian tube and Sertoli cells in testis also stain strongly (HPA tissue IHC). HPA rates the tissue pattern Supported, with medium consistency between staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Granular cytoplasmic staining in glandular cells, with stronger ciliary-rootlet or Sertoli-cell staining in the relevant sections.	This fits the observed AGPS tissue pattern (HPA tissue IHC). Score the named cell population and its compartment separately: HPA reports High staining in bronchial and fallopian-tube ciliated cells and testicular Sertoli cells, and Medium staining in several glandular populations (HPA tissue IHC).
Predominantly nuclear staining, or a smooth membrane outline without discernible cytoplasmic granules.	That distribution does not match HPA's granular cytoplasmic IHC pattern or its peroxisomal ICC-IF localization (HPA tissue IHC; HPA subcellular ICC-IF). Treat it as a possible artifact and compare with section morphology and detection controls (general IHC practice). Chromogenic granules alone do not prove individual peroxisomes.
Strong staining in adipocytes, alveolar cells, cardiomyocytes, or hematopoietic cells.	HPA lists these specific populations as Not detected, despite describing AGPS staining overall as ubiquitous (HPA tissue IHC). Recheck cell identity before calling the result AGPS-positive; cross-reactivity or endogenous detection activity is possible (general IHC practice). A different cell type in the same tissue needs its own comparison.
Diffuse color across cells and surrounding tissue obscures the expected granules.	The slide cannot support compartment-based scoring against HPA's granular cytoplasmic pattern (HPA tissue IHC). Excess detection background, insufficient blocking, or excess primary antibody are possible general IHC causes; this observation does not establish an AGPS-specific fixation effect (general IHC practice).
No signal in the relevant cells of bronchus, fallopian tube, or testis.	HPA reports High staining in ciliated cells of bronchus and fallopian tube and in testicular Sertoli cells (HPA tissue IHC). Check that the expected cells are present, then review the assay controls and IHC-validated antibody before interpreting the absent signal as biological loss (general IHC practice).

💡Expected AGPS appearanceA credible positive is granular cytoplasmic color in the expected cells, with High staining in the named ciliated or Sertoli cells and Medium staining in listed glandular cells; dominant nuclear color or uniform diffuse staining is discordant (HPA tissue IHC; HPA subcellular ICC-IF).

How each factor affects the staining
Compartment and visual resolution	UniProt places AGPS in peroxisomes and at their membrane, without a transmembrane segment (UniProt O00116). HPA reports granular cytoplasmic tissue staining and enhanced peroxisomal ICC-IF localization (HPA tissue IHC; HPA subcellular ICC-IF). Use the granules as an IHC pattern, not as proof that each visible dot is a peroxisome (general IHC practice).
Cell population and evidence strength	HPA describes low tissue RNA specificity and a ubiquitous granular protein pattern, while listing distinct High, Medium, Low, and Not detected cell populations (HPA tissue IHC). Its Supported rating has medium staining–RNA consistency (HPA tissue IHC); compare like cell populations rather than treating a whole organ as uniformly positive or negative.
Antibody validation	HPA rates IHC for HPA030209, HPA030210, and HPA030211 as Supported (HPA antibodies). HPA rates ICC for HPA030209 and HPA030210 as Enhanced (HPA antibodies). These ratings support checking assay-specific evidence; ICC localization does not by itself validate a paraffin-section result.
Processing and epitope uncertainty	UniProt records a processed AGPS chain spanning residues 59–658, with no annotated signal peptide, propeptide, or transmembrane segment (UniProt O00116). The supplied record gives no antibody epitope, so it cannot predict whether processing changes staining with the chosen IHC-validated antibody. Target-specific fixation sensitivity is unreported here.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Known-positive cells show no chromogenic signal.	The expected population may be absent from the section, or the IHC detection workflow may have failed; HPA reports High staining only for specified cells in bronchus, fallopian tube, and testis (HPA tissue IHC).	Identify those cells on the counterstained section, inspect a positive control, and check the IHC-validated antibody and detection reagents (general IHC practice). Review retrieval conditions as a general workflow check; no AGPS-specific retrieval requirement is supplied.
Nuclei dominate while cytoplasmic granules are faint or absent.	The distribution conflicts with HPA tissue IHC and peroxisomal ICC-IF localization (HPA tissue IHC; HPA subcellular ICC-IF). Nonspecific staining is possible (general IHC practice).	Compare the same cell type across sections, review the no-primary control, and reassess primary-antibody concentration and detection background (general IHC practice).
A listed HPA-negative cell population stains strongly.	HPA reports Not detected for specified cells, including alveolar cells and cardiomyocytes (HPA tissue IHC). Misidentification, cross-reactivity, or endogenous detection activity may explain a discrepancy (general IHC practice).	Confirm cell identity, compare a no-primary control, and use an independent IHC-validated antibody if available (general IHC practice). Report the observed population rather than labeling the entire tissue discordant.
Diffuse color prevents granular scoring.	Background can obscure HPA's granular cytoplasmic pattern (HPA tissue IHC). Excess primary antibody, incomplete blocking, or endogenous detection activity are possible workflow causes (general IHC practice).	Review blocking and detection controls; titrate the primary antibody within its validated IHC conditions (general IHC practice). Do not infer an AGPS-specific fixation problem from this appearance alone.
A low-staining population is mistaken for a failed positive control.	HPA lists neuronal cells of cerebral cortex, hepatocytes, and pancreatic exocrine glandular cells as Low, while naming stronger populations elsewhere (HPA tissue IHC).	Use the appropriate named cell population for the comparison and record intensity by cell type (HPA tissue IHC; general IHC practice). A low HPA category should not be used as the sole strong-signal benchmark.
Can IF/ICC clarify whether the granular signal is peroxisomal?	HPA reports enhanced peroxisomal ICC-IF localization, while chromogenic tissue IHC is described as granular cytoplasmic staining (HPA subcellular ICC-IF; HPA tissue IHC).	Consult the separate IF/ICC guide for that application. Its localization evidence can inform interpretation, but the paraffin-section IHC result must be judged against the tissue pattern and IHC validation (HPA tissue IHC; HPA antibodies).

Source: UniProt O00116 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing AGPS staining

IHC analysis of AGPS using anti-AGPS antibody (A03481-1). AGPS was detected in a paraffin-embedded section of human adenocarcinoma of lung 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-AGPS Antibody (A03481-1) 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.

A03481-1 · IHCIHC analysis of AGPS using anti-AGPS antibody (A03481-1). AGPS was detected in a paraffin-embedded section of human adenocarcinoma of lung t

IF analysis of AGPS using anti-AGPS antibody (A03481-1). AGPS was detected in an immunocytochemical section of SiHa 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 5 μg/mL rabbit anti-AGPS Antibody (A03481-1) 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.

A03481-1 · IFIF analysis of AGPS using anti-AGPS antibody (A03481-1). AGPS was detected in an immunocytochemical section of SiHa cells. Enzyme antigen re

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000018510-AGPS/tissue/cerebral+cortex#imid_8049699

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000018510-AGPS/tissue/colon#imid_8042057

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000018510-AGPS/tissue/liver#imid_8042058

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000018510-AGPS/tissue/kidney#imid_8042059

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA030209; green = AGPS, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for AGPS IHC & IF

🧪Run bronchus first and look for staining in ciliated-cell rootlets (HPA: High in bronchus ciliated cells). Use adipose tissue as the negative tissue (HPA: Not detected in adipocytes), and compare rootlet staining with adjacent cells and regions lacking rootlet signal on the positive slide without assuming those cells are AGPS-negative.

Positive control tissue: Bronchus (Ciliated cells (ciliary rootlets), HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show AGPS in A-431, U-251MG, U2OS, with annotated localisation: Peroxisomes (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a matched rabbit IgG isotype control (caption: rabbit anti-AGPS primary), plus AGPS-knockout material as a biological negative where available. Quench endogenous peroxidase before HRP/DAB detection and check whether the bronchial apical signal persists in the no-primary control (caption: HRP/DAB detection; HPA: bronchial ciliary-rootlet staining).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected A03481-1 paraffin-section caption does not state a fixative (caption: fixative unreported). Heat retrieval with EDTA at pH 8.0 was used, but its necessity is untested in the supplied evidence (caption: EDTA pH 8.0). Frozen sections are not established as easier; IF/ICC offers a peroxisomal localization check in the listed cell lines (HPA: peroxisomes enhanced), while bronchial rootlet signal should be distinguished from apical background (HPA: High in bronchial ciliated-cell rootlets).

HPA tissue IHC evidence for AGPS

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Supported — 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
Bronchus	Ciliated cells (ciliary rootlets)	High	Protein (IHC)	HPA →
Fallopian tube	Ciliated cells (ciliary rootlets)	High	Protein (IHC)	HPA →
Testis	Sertoli cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	Medium	Protein (IHC)	HPA →
Cerebellum	Purkinje 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 →
Bone marrow	Hematopoietic cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced AGPS IHC Tips

Troubleshoot AGPS staining in paraffin sections using the catalog antibody’s IHC example, peroxisomal localisation, and cell-specific tissue controls.

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

Use heat-mediated retrieval in EDTA at pH 8.0 for AGPS paraffin-section IHC (datasheet A03481-1). The selected example used 2 μg/ml primary antibody overnight at 4°C after blocking with 10% goat serum; hold those conditions steady while comparing retrieval heating times on serial sections (caption A03481-1; standard IHC practice). The caption gives no heating duration, so record the time and cooling conditions for each comparison (caption A03481-1; standard IHC practice). Check whether granular cytoplasmic staining improves in glandular cells without a parallel rise in diffuse background, using a no-primary control to assess detection-related signal (HPA: tissue profile; standard IHC practice).

Could fixation explain weak or uneven AGPS staining?

The selected paraffin-section caption does not state a fixative, so AGPS-specific fixation sensitivity and an optimal fixation duration are unknown (caption A03481-1). Record each specimen’s fixative and fixation time, then compare similarly processed sections before attributing a staining difference to AGPS abundance (standard IHC practice). Use the documented EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody overnight at 4°C as consistent starting conditions (datasheet A03481-1; caption A03481-1). Evaluate preserved morphology and granular cytoplasmic signal together, and do not infer a fixation effect from the protein’s peroxisomal localisation or modifications (HPA: tissue profile; UniProt O00116; standard IHC practice).

What staining pattern should count as plausible AGPS localisation?

Expect predominantly granular cytoplasmic staining in tissue IHC, consistent with the reported peroxisomal localisation of AGPS (HPA: tissue profile and subcellular localisation; UniProt O00116). Assess the pattern within identified cell types: HPA reports high staining in bronchial ciliated cells at ciliary rootlets and in testicular Sertoli cells, with medium staining in colon glandular cells (HPA: tissue IHC). The record lists no transmembrane segment, so a continuous cell-surface rim is not the expected pattern from its annotated topology (UniProt O00116 topology; standard IHC interpretation). Compare suspicious nuclear or diffuse staining with no-primary controls and the surrounding morphology before calling cells positive (standard IHC practice).

How could epitope placement affect AGPS IHC interpretation?

Confirm the catalog antibody’s immunogen or mapped epitope before interpreting regional loss of staining, because no epitope position is supplied here (standard IHC practice). The record lists no isoforms and annotates a processed chain spanning residues 59–658; it also places a FAD-binding domain at residues 202–384 (UniProt O00116). Annotated modifications include phosphoserine at 65, phosphothreonine at 74, and acetyllysines at 102 and 347, but their effect on this antibody’s binding is unknown (UniProt O00116). If sections disagree, compare retrieval and tissue processing first, then use an independently mapped epitope as an orthogonal check (standard IHC practice).

How should I investigate AGPS localisation by IF alongside the IHC result?

For exploratory IF, pair AGPS with a marker identifying the expected cell type, such as ciliated cells in bronchial tissue, and consider a peroxisomal marker to assess punctate overlap (HPA: tissue IHC and subcellular localisation; standard IF practice). Select spectrally separated fluorophores after checking tissue autofluorescence in unstained controls, and keep acquisition settings consistent across comparisons (standard IF practice). AGPS has no annotated transmembrane segment, but this antibody’s epitope position and membrane accessibility are unspecified; optimise permeabilisation empirically for the epitope’s location (UniProt O00116 topology; standard IF practice). The paraffin-section IHC example supplies no IF fixation or permeabilisation conditions, so validate IF staining with appropriate controls (caption A03481-1; standard IF practice).

How can I reduce diffuse brown signal without losing AGPS staining?

First compare a no-primary section with the test section to separate detection-system background from primary-dependent staining (standard IHC practice). Block endogenous peroxidase before HRP and DAB development, and inspect tissue edges and damaged regions where nonspecific chromogen deposition can complicate reading (standard IHC practice). The selected example used 10% goat serum, 2 μg/ml rabbit primary overnight at 4°C, and a peroxidase-conjugated secondary for 30 minutes at 37°C (caption A03481-1). If background persists, optimise blocking, washing and primary concentration on serial sections while retaining EDTA retrieval at pH 8.0 as the documented starting point (datasheet A03481-1; standard IHC practice).

How should AGPS staining be scored across heterogeneous tissue? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and region before scoring, because AGPS staining varies among annotated tissue cell types (HPA: tissue IHC; standard IHC practice). For chromogenic IHC, report the percentage of positive cells and an intensity-based H-score, or count positive cells per mm² when discrete populations are being compared (standard IHC practice). Normalise counts to the relevant intact tissue area or cell population, and apply the same positivity threshold, counterstain and image settings to every section (standard IHC practice). Score granular cytoplasmic signal separately from diffuse deposit, then document retrieval and antibody conditions so technical differences are visible (HPA: tissue profile; standard IHC practice).

When is an apparent AGPS-positive result likely to be artefactual?

Treat granular cytoplasmic staining in an anatomically plausible cell population as stronger evidence than isolated nuclear, uniform membrane-rim or diffuse extracellular colour (HPA: tissue profile and subcellular localisation; UniProt O00116 topology; standard IHC interpretation). Check whether signal clusters at cut edges, necrotic regions or damaged tissue, and whether it persists without primary antibody or after peroxidase blocking (standard IHC practice). Cell identity matters: HPA reports high bronchial ciliated-cell staining but no detectable staining in lung alveolar cells, while the selected antibody caption shows a lung adenocarcinoma section (HPA: tissue IHC; caption A03481-1). Interpret discordant cases cautiously because HPA rates tissue staining as supported with medium RNA–protein consistency (HPA: reliability).

Boster reagents

Best AGPS / Alkyldihydroxyacetonephosphate synthase, peroxisomal IHC Antibodies

The catalog antibody has IHC data from human paraffin sections of lung adenocarcinoma, cervical cancer and esophageal squamous cell carcinoma, plus IF data from SiHa cells (catalog image captions).

Real IHC data

Anti-AGPS Antibody ®

Cat # A03481-1

View product →

A03481-1 is listed for human IHC and is shown on paraffin sections of lung adenocarcinoma, cervical cancer and esophageal squamous cell carcinoma (catalog applications and IHC image captions). A03481-1 is also listed for ICC/IF and is shown in SiHa cells (catalog applications and IF image caption).

Which to pick: Choose A03481-1 for human paraffin-section IHC; its IHC captions report 2 μg/ml antibody after EDTA pH 8.0 retrieval, but do not report the fixative (catalog IHC image captions). Choose A03481-1 for ICC/IF in SiHa cells, where its IF caption reports 5 μg/ml antibody (catalog IF image caption). No cross-species choice is supported: A03481-1 lists Human reactivity only, and its clonality is unreported (catalog reactivity and clone fields).

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

References

UniProt Consortium. UniProt entry O00116 (ADAS_HUMAN, Alkyldihydroxyacetonephosphate synthase, peroxisomal).
Human Protein Atlas. AGPS tissue IHC expression (reliability: Supported).
Human Protein Atlas. AGPS subcellular location (ICC-IF): Localized to the peroxisomes..
Human Protein Atlas. AGPS antibody validation summary (3 antibodies).
Multiple oestradiol functions inhibit ferroptosis and acute kidney injury. Nature 2025 — PMC12460175.
Alkylglycerone phosphate synthase (AGPS) deficient mice: models for rhizomelic chondrodysplasia punctate type 3 (RCDP3) malformation syndrome. Molecular genetics and metabolism reports 2014 — PMC4151185.
TrkA promotes MDM2-mediated AGPS ubiquitination and degradation to trigger prostate cancer progression. Journal of experimental & clinical cancer research : CR 2024 — PMC10782585.
PubMed PMID:9187299 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.