ALDH6A1 / Methylmalonate-semialdehyde/malonate-semialdehyde dehydrogenase [acylating], mitochondrial · IHC design guide

Design Immunohistochemistry for ALDH6A1

Use granular cytoplasmic staining in renal tubules and hepatocytes as the tissue reference for ALDH6A1 chromogenic IHC (HPA tissue IHC). This guide covers paraffin section controls, antibody dilution and scoring by cell type and staining pattern (standard IHC practice).

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

Immunohistochemistry protocol sheet for ALDH6A1 (IHC for ALDH6A1): expected localisation Observed granular cytoplasm (HPA tissue IHC); mitochondrial protein (UniProt), antibody A08868-1, validated IHC image, and IHC protocol steps — Printable **ALDH6A1** IHC protocol sheet — expected localisation Observed granular cytoplasm (HPA tissue IHC); mitochondrial protein (UniProt), antibody A08868-1, controls and protocol steps. Open the full ALDH6A1 IHC guide →

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ALDH6A1 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	Observed granular cytoplasm (HPA tissue IHC); mitochondrial protein (UniProt)
	Staining pattern	Renal tubules and hepatocytes: granular cytoplasmic staining (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A08868-1)
	Positive control ⓘ	Caudate+4 more · see all
	Negative control ⓘ	Adipose tissue+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation consistent across sections (standard IHC practice; not target-specific)
	Caveat	Staining has medium consistency with RNA expression (HPA tissue IHC)
	Regulation	No expression regulator annotated (UniProt)
	Isoform / epitope	2 isoforms; epitope coverage unspecified (UniProt)

Section 1

Recommended ALDH6A1 IHC & IF Protocols

Compare the catalog antibody’s IHC-P protocol (datasheet) with published ALDH6A1 staining of mouse embryo sections (PMC13509631) and renal tissue sections (PMC7038561).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human liver cancer tissue; fixative not specified (datasheet A08868-1)
Fixation	Image fixative and duration unreported (datasheet A08868-1); 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 A08868-1); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A08868-1)
Primary antibody	Rabbit anti-ALDH6A1, 2μg/ml (datasheet A08868-1)
Primary incubation	Overnight at 4 °C (datasheet A08868-1)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A08868-1)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	ALDH6A1-positive staining in glial cells of caudate (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in renal tubules and hepatocytes. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA pH 8.0 retrieval for the catalog antibody (datasheet: A08868-1); neither article specifies a retrieval buffer (PMC13509631; PMC7038561).

Section 2

What Is the Expected ALDH6A1 Staining Pattern?

ALDH6A1 is a mitochondrial protein with no transmembrane segment (UniProt Q02252: subcellular location and topology). In paraffin section IHC, expect granular cytoplasmic staining in renal tubules and hepatocytes (HPA: tissue IHC profile). HPA rates the tissue staining profile Enhanced, while reporting medium consistency between antibody staining and RNA expression; interpret intensity alongside the cell type and compartment (HPA: tissue IHC reliability).

What am I looking at on my slide?
Granular cytoplasmic chromogen appears in renal tubules and hepatocytes (HPA: tissue IHC profile).	This matches the observed tissue pattern and mitochondrial location (HPA: tissue IHC profile; UniProt Q02252: subcellular location). Granules are compatible with mitochondrial staining, but chromogenic IHC alone cannot establish organelle colocalisation (general IHC practice).
Signal is chiefly nuclear or outlines cell surfaces instead of forming cytoplasmic granules.	Treat that compartment pattern as suspect: UniProt places ALDH6A1 in mitochondria and reports no transmembrane segment (UniProt Q02252: location and topology). Check morphology and controls before interpreting it as ALDH6A1; an unexpected compartment does not identify the artefact's cause (general IHC practice).
Strong staining appears in adipocytes, alveolar cells or skeletal muscle myocytes (HPA: not detected in these cells).	These are poor matches to the reported IHC pattern (HPA: tissue IHC). Consider antibody cross-reactivity or endogenous detection activity, particularly if the signal persists in a no-primary control; that control helps distinguish detection background from primary-antibody staining (general IHC practice).
Chromogen spreads evenly across tissue, including spaces between cells, rather than marking cytoplasmic granules.	A diffuse deposit is difficult to score as ALDH6A1 because the reported pattern is granular and cytoplasmic (HPA: tissue IHC profile). Review the no-primary control and detection background, then optimise blocking, washes and chromogen development as general IHC practice.
No signal is visible in renal tubules or hepatocytes on an otherwise interpretable section.	The result conflicts with the reported positive pattern (HPA: tissue IHC profile), but a blank slide alone cannot distinguish absent antigen from assay failure (general IHC practice). Confirm tissue preservation and counterstain, then check the catalog antibody's validated IHC conditions and a concurrent positive control.

💡Expected ALDH6A1 appearanceCall the result pattern-consistent when renal tubules and hepatocytes show detectable granular cytoplasmic staining (HPA: tissue IHC profile), with intensity assessed against controls; predominantly nuclear, surface-only or diffuse extracellular colour is suspect for this mitochondrial target (UniProt Q02252: location and topology; general IHC practice).

How each factor affects the staining
Cell type and tissue context	HPA describes granular cytoplasmic expression in renal tubules and hepatocytes, while its RNA category is group enriched in kidney and liver (HPA: tissue IHC profile and RNA specificity). Use the protein staining pattern to judge an IHC slide; the RNA category does not assign a chromogen intensity to either cell type.
Reported negative and high-staining cells	HPA reports no detection in adipocytes and alveolar cells, but high staining in glial cells of caudate and cerebral cortex (HPA: tissue IHC). Score the identified cells rather than treating every cell in a tissue as uniformly positive or negative (general IHC practice).
Evidence strength	HPA labels the tissue IHC profile Enhanced but describes medium consistency with RNA expression (HPA: tissue IHC reliability). Its listed antibodies HPA029072, HPA029073, HPA029074 and HPA029075 each have Enhanced IHC status (HPA: antibody validation); this supports the reported pattern without validating every new staining run.
Protein processing and isoforms	UniProt lists two isoforms and a mature chain spanning residues 34–535 (UniProt Q02252: isoforms and processing). The payload gives no antibody epitope, so these annotations cannot predict whether a given antibody distinguishes isoforms or recognises the processed protein in sections.
IF/ICC Q: Where should ALDH6A1 fluorescence appear?	A: Mainly in mitochondria (HPA: subcellular ICC-IF, enhanced; UniProt Q02252: location). This is the IF/ICC expectation; use that application's own guide for its staining workflow.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Renal tubules and hepatocytes are both blank (HPA: expected granular cytoplasmic expression).	The assay may have failed; the blank result does not by itself identify the failed step (general IHC practice).	Run a concurrent positive section and inspect counterstain, antibody use, retrieval and detection steps against the catalog antibody's IHC-P instructions (general IHC practice).
Signal is mainly nuclear or confined to cell borders.	That distribution conflicts with mitochondrial ALDH6A1 and its lack of a transmembrane segment (UniProt Q02252: location and topology).	Recheck cell boundaries and the no-primary control; compare a repeat section with the reported granular cytoplasmic pattern (HPA: tissue IHC; general IHC practice).
Adipocytes or alveolar cells stain strongly (HPA: not detected in these cells).	Possible cross-reactivity or endogenous detection activity; the slide alone cannot separate them (general IHC practice).	Compare no-primary and positive-tissue controls, then review blocking and the detection system for background (general IHC practice).
Broad, diffuse colour obscures individual cells and granules.	Nonspecific background or excessive chromogen development can obscure a granular cytoplasmic pattern (general IHC practice; HPA: tissue IHC profile).	Check the no-primary control, washing and chromogen development, and rescore only where cell morphology remains clear (general IHC practice).
The expected cells stain, but only weakly.	HPA reports the pattern without assigning a universal intensity threshold to renal tubules or hepatocytes (HPA: tissue IHC profile).	Compare matched positive and no-primary controls before changing interpretation; follow the catalog antibody's IHC-P conditions when adjusting the assay (general IHC practice).
A new section differs from the HPA image pattern.	HPA's tissue profile has medium consistency with RNA expression; a mismatch needs review rather than an automatic biological explanation (HPA: reliability).	Verify the scored cell type and cytoplasmic distribution, then compare an independent IHC-validated antibody if available (HPA: antibody validation; general IHC practice).

Source: UniProt Q02252 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing ALDH6A1 staining

IHC analysis of ALDH6A1 using anti-ALDH6A1 antibody (A08868-1). ALDH6A1 was detected in paraffin-embedded section of human liver 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 2μg/ml rabbit anti-ALDH6A1 Antibody (A08868-1) 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.

A08868-1 · IHCIHC analysis of ALDH6A1 using anti-ALDH6A1 antibody (A08868-1). ALDH6A1 was detected in paraffin-embedded section of human liver cancer tiss

Immunohistochemical analysis of paraffin-embedded H.colon section using ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. A peroxidase-conjugated goat anti-mouse IgG at 1:400 dilution was used as the secondary antibody, followed by DAB staining.

M08868 · IHCImmunohistochemical analysis of paraffin-embedded H.colon section using ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. A peroxidase-

Immunohistochemical analysis of paraffin-embedded H.liver section using ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. A peroxidase-conjugated goat anti-mouse IgG at 1:400 dilution was used as the secondary antibody, followed by DAB staining.

M08868 · IHCImmunohistochemical analysis of paraffin-embedded H.liver section using ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. A peroxidase-

Fluorescent image of MCF-7 cells stained with ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. An Alexa Fluor® 488-conjugated goat anti-mouse lgG at 1:400 dilution was used as the secondary antibody (green). DAPI was used to stain the cell nuclear (blue). Cytoplasmic actin was counterstained with Alexa Fluor® 555 conjugated with Phalloidin (red).

M08868 · IFFluorescent image of MCF-7 cells stained with ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. An Alexa Fluor® 488-conjugated goat ant

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000119711-ALDH6A1/tissue/cerebral+cortex#imid_7909248

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000119711-ALDH6A1/tissue/colon#imid_7906950

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000119711-ALDH6A1/tissue/liver#imid_7906959

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000119711-ALDH6A1/tissue/kidney#imid_7906952

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA029073; green = ALDH6A1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for ALDH6A1 IHC & IF

🧪Run cerebellum first and score staining in Bergmann glia (HPA: High in Bergmann glia). Run adipose tissue as a negative comparator (HPA: Not detected in adipocytes); on the cerebellum slide, compare Bergmann glia with neighboring cells, but do not assume those cells are ALDH6A1-negative from the supplied HPA rows.

Positive control tissue: Caudate (Glial cells, HPA High)

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show ALDH6A1 in A-431, U-251MG, U2OS, MCF-7, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a host- and clonality-matched rabbit isotype control (hero caption: rabbit primary; standard IHC practice). Use an ALDH6A1 knockout specimen as a biological negative where available, and check for endogenous biotin before using the caption’s avidin–biotin detection system (hero caption: SABC detection; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the selected A08868-1 caption does not state a fixative (hero caption: fixative not stated). The caption uses heat retrieval in EDTA at pH 8.0, but does not establish that retrieval is required (hero caption: EDTA heat retrieval). Frozen-section performance and relative ease are unreported; ICC-IF has mitochondrial localization evidence, while cerebellar autofluorescence should be assessed if using IF (HPA subcellular: mitochondria, ICC-IF images available; standard IF practice).

HPA tissue IHC evidence for ALDH6A1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — 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
Caudate	Glial cells	High	Protein (IHC)	HPA →
Cerebellum	Bergmann glia - cytoplasm/membrane	High	Protein (IHC)	HPA →
Cerebral cortex	Glial cells	High	Protein (IHC)	HPA →
Cervix	Glandular cells	High	Protein (IHC)	HPA →
Duodenum	Glandular cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Lung	Alveolar cells	Not detected	Protein (IHC)	HPA →
Nasopharynx	Respiratory epithelial cells	Not detected	Protein (IHC)	HPA →
Oral mucosa	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →
Placenta	Decidual cells	Not detected	Protein (IHC)	HPA →

Section 3

Advanced ALDH6A1 IHC Tips

Troubleshoot ALDH6A1 staining in paraffin sections by checking retrieval, cytoplasmic localisation, cell identity and detection controls before comparing signal intensity (UniProt Q02252; HPA tissue IHC).

What should I change when ALDH6A1 staining is weak after antigen retrieval?

Start with heat-mediated retrieval in EDTA at pH 8.0 for the catalog antibody (datasheet A08868-1). Its paraffin-section example used this retrieval before staining human liver cancer tissue, but the caption does not state the fixative (datasheet A08868-1). If signal remains weak, compare retrieval intensity on matched sections while keeping antibody concentration, detection and development consistent (standard IHC practice). Check whether renal tubules or hepatocytes show granular cytoplasmic staining before treating a faint result as target absence (HPA tissue IHC). Excessive retrieval can damage tissue morphology, so judge staining together with section integrity and the negative control (standard IHC practice).

Could fixation explain weak or uneven ALDH6A1 staining?

Target-specific fixation sensitivity for ALDH6A1 is unknown from the supplied evidence; the catalog example identifies a paraffin section but does not report its fixative (datasheet A08868-1). Record fixation conditions for each specimen and compare sections processed under consistent conditions before attributing signal differences to biology (standard IHC practice). Examine morphology, section adhesion and the distribution of staining across the section when investigating uneven results (standard IHC practice). If fixation histories differ, interpret intensity comparisons cautiously and use matched processing where possible (standard IHC practice). Neither the reported tissue pattern nor mitochondrial localisation establishes a particular fixation effect for this antibody (HPA tissue IHC; UniProt Q02252).

How should I assess cytoplasmic versus nuclear ALDH6A1 staining?

Expect a granular cytoplasmic pattern in renal tubules and hepatocytes, consistent with the reported mitochondrial localisation of ALDH6A1 (HPA tissue IHC; UniProt Q02252). Evaluate chromogen against a light counterstain at sufficient magnification to distinguish intracellular granules from diffuse cytoplasmic or nuclear colour (standard IHC practice). Strong nuclear staining without the expected cytoplasmic pattern deserves comparison with a no-primary control and a positive tissue region (HPA tissue IHC; standard IHC practice). The protein has no annotated transmembrane segment, so a crisp cell-surface outline alone would be unexpected (UniProt Q02252). Preserve cell boundaries and morphology when judging whether apparent granules belong to the target cells (standard IHC practice).

Can isoforms or mitochondrial processing change what this antibody detects in sections?

ALDH6A1 has 2 listed isoforms, and its annotated mature chain spans residues 34–535 (UniProt Q02252). The supplied caption does not identify the antibody epitope, so its recognition of each isoform or processed form cannot be established here (datasheet A08868-1; UniProt Q02252). Compare staining across identically processed sections and use an independent antibody with a documented epitope if isoform discrimination matters (standard IHC practice). Several modified residues are annotated, including acetylated and succinylated lysines near the amino terminus, but their effect on this antibody is unknown (UniProt Q02252). Do not infer an isoform-specific distribution from chromogenic intensity alone (standard IHC practice).

How can IF help investigate ambiguous ALDH6A1 IHC localisation?

Use IF as a separate localisation check, pairing ALDH6A1 with a mitochondrial marker and a marker that identifies the expected cell type (HPA subcellular; standard IF practice). For tissue comparison, renal tubules or hepatocytes provide documented granular cytoplasmic IHC patterns (HPA tissue IHC). Choose spectrally separated fluorophores after checking tissue autofluorescence, and include single-label and no-primary controls to assess bleed-through and background (standard IF practice). ALDH6A1 is mitochondrial and has no annotated transmembrane segment; permeabilisation should therefore allow antibody access to the intracellular epitope, whose exact position is unreported here (UniProt Q02252; standard IF practice). Optimise IF staining independently of the paraffin-section IHC example (datasheet A08868-1; standard IF practice).

How do I reduce diffuse brown background without losing ALDH6A1 signal?

First compare a no-primary section with the test section to separate detection background from antibody-dependent staining (standard IHC practice). The catalog example used 10% goat serum blocking, 2 μg/mL primary antibody overnight at 4°C, and a biotin-based DAB detection system (datasheet A08868-1). Ensure adequate washing and peroxidase blocking, then inspect whether background tracks tissue edges, damaged areas or endogenous pigment (standard IHC practice). With biotin-based detection, assess endogenous biotin as another possible source of signal (standard IHC practice; datasheet A08868-1). Retain granular cytoplasmic staining in expected cells as the reference pattern while adjusting blocking or detection (HPA tissue IHC; standard IHC practice).

What is a defensible way to quantify ALDH6A1 IHC across specimens? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue compartment before scoring, because ALDH6A1 staining varies by cell type in the supplied tissue profile (HPA tissue IHC). Score the percentage of positive cells and staining intensity, or calculate an H-score on the standard 0–300 scale using consistent thresholds (standard IHC practice). Normalise counts to eligible cells, or stained area to viable tissue area, and report the chosen denominator for each specimen (standard IHC practice). Exclude folds, edges, necrosis and poorly preserved regions using the same criteria across sections (standard IHC practice). Keep retrieval, antibody concentration, DAB development and imaging conditions consistent when comparing intensity (standard IHC practice).

How can I distinguish true ALDH6A1 staining from artefact?

A plausible positive shows granular cytoplasmic staining in the relevant cells, matching the renal-tubule and hepatocyte pattern and mitochondrial localisation (HPA tissue IHC; HPA subcellular). Check cell identity carefully: the supplied profile also reports high staining in several glial and glandular cell populations, while some other populations are not detected (HPA tissue IHC). Predominantly nuclear colour, staining confined to cut edges or necrotic regions, and signal persisting without primary antibody warrant investigation as artefacts (UniProt Q02252; standard IHC practice). Control for endogenous peroxidase and, with the catalog biotin-based system, endogenous biotin before assigning brown deposits to ALDH6A1 (datasheet A08868-1; standard IHC practice).

Boster reagents

Best ALDH6A1 / Methylmalonate-semialdehyde/malonate-semialdehyde dehydrogenase [acylating], mitochondrial IHC Antibodies

ALDH6A1 antibodies have IHC images from human paraffin sections and an IF image from MCF-7 cells (catalog image captions). Catalog reactivity covers human, mouse, and rat (catalog reactivity).

Real IHC data

Anti-ALDH6A1 Antibody ®

Cat # A08868-1

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Real IHC data Immunohistochemical analysis of paraffin-embedded H.colon section using ALDH6A1 Antibody. M08868 was diluted at 1:25 dilution. A peroxidase-conjugated goat anti-mouse IgG at 1:400 dilution was used as the secondary antibody, followed by DAB staining.

Anti-ALDH6A1 Antibody

Cat # M08868

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A08868-1 has an IHC image from a paraffin section of human liver cancer tissue (A08868-1 image caption). M08868 has IHC images from human colon and liver paraffin sections and an IF image from MCF-7 cells (M08868 image captions).

Which to pick: For tissue IHC, A08868-1 is a rabbit polyclonal option shown on a human liver cancer paraffin section, while M08868 is a mouse monoclonal option shown on human colon and liver paraffin sections at 1:25; the captions do not report a fixative (catalog antibody details; A08868-1 and M08868 IHC captions). For IF/ICC, choose M08868: IF is listed and its image shows MCF-7 cells stained at 1:25 (M08868 applications and IF caption). For work involving mouse or rat samples, A08868-1 lists reactivity with both, but its stated paraffin-section IHC testing and image are human only (A08868-1 catalog reactivity, dilution notes, and IHC caption).

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

References

UniProt Consortium. UniProt entry Q02252 (MMSA_HUMAN, Methylmalonate-semialdehyde/malonate-semialdehyde dehydrogenase [acylating], mitochondrial).
Human Protein Atlas. ALDH6A1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. ALDH6A1 subcellular location (ICC-IF): Mainly localized to the mitochondria..
Human Protein Atlas. ALDH6A1 antibody validation summary (4 antibodies).
Cross-Species Conservation and Function of ALDH6A1/Aldh6a1 Validate Zebrafish and Mouse as Complementary Experimental Systems for Methylmalonate Semialdehyde Dehydrogenase Deficiency. Biology 2026 — PMC13509631.
ABAT and ALDH6A1, regulated by transcription factor HNF4A, suppress tumorigenic capability in clear cell renal cell carcinoma. Journal of translational medicine 2020 — PMC7038561.
The downregulated drug-metabolism related ALDH6A1 serves as predictor for prognosis and therapeutic immune response in gastric cancer. Aging 2022 — PMC9512493.
PubMed PMID:10947204 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:12508121 — UniProt-cited evidence.