TRAP1 / Heat shock protein 75 kDa, mitochondrial · IHC design guide

Design Immunohistochemistry for TRAP1

Plan TRAP1 staining in paraffin sections around its granular cytoplasmic tissue pattern (HPA tissue IHC). The guide uses the catalog antibody’s documented 1 μg/ml IHC example and highlights cell populations that can affect interpretation (datasheet PB9834; HPA tissue IHC).

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

Immunohistochemistry protocol sheet for TRAP1 (IHC for TRAP1): expected localisation Granular cytoplasm in tissue sections (HPA tissue IHC), antibody PB9834, validated IHC image, and IHC protocol steps — Printable **TRAP1** IHC protocol sheet — expected localisation Granular cytoplasm in tissue sections (HPA tissue IHC), antibody PB9834, controls and protocol steps. Open the full TRAP1 IHC guide →

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TRAP1 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 sections (HPA tissue IHC)
	Staining pattern	Granular cytoplasmic staining across tissues (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet PB9834)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 44 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Matched tissue-IHC evidence does not establish the fixation claim. Validate the specimen-specific method before use. (selected-SKU IHC image PB9834)
	Caveat	Adipocytes and hematopoietic cells can stain strongly (HPA tissue IHC)
	Regulation	Reduced in bladder and renal cancers (UniProt)
	Isoform / epitope	2 isoforms; mature chain starts at residue 60; epitope differences unknown (UniProt)

Section 1

Recommended TRAP1 IHC & IF Protocols

The catalog antibody’s IHC-P protocol uses EDTA pH 8.0 heat retrieval (datasheet: PB9834). The published TRAP1 IHC protocols below cover ovarian, colorectal, lung and breast cancer specimens (PMC3533746; PMC5400579; PMC5207305; PMC4792571).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human intestinal cancer tissue; fixative not specified (datasheet PB9834)
Fixation	Image fixative and duration unreported (datasheet PB9834); 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 PB9834); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet PB9834)
Primary antibody	Rabbit anti-TRAP1, 0.5-1μg/ml (datasheet PB9834)
Primary incubation	Overnight at 4 °C (datasheet PB9834)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet PB9834)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	TRAP1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Granular cytoplasmic expression in all tissues. No signal in the no-primary control.

💡Decision noteStart with EDTA pH 8.0 heat retrieval for the catalog antibody (datasheet: PB9834); citrate pH 6 pressure retrieval was reported with a different antibody (PMC5207305).

Section 2

What Is the Expected TRAP1 Staining Pattern?

TRAP1 should produce granular cytoplasmic IHC staining, consistent with its mitochondrial matrix and inner membrane localisation and absence of a transmembrane segment (HPA tissue IHC; UniProt Q12931 topology). HPA reports staining across tissues, including high staining in adrenal glandular cells, bronchial respiratory epithelial cells and bone marrow hematopoietic cells (HPA tissue IHC). The tissue profile has Enhanced reliability, reflecting consistency between antibody staining and RNA expression (HPA tissue IHC).

What am I looking at on my slide?
Granular cytoplasmic signal in an expected cell population.	This fits the reported tissue pattern and mitochondrial localisation (HPA tissue IHC; UniProt Q12931). Judge the cellular pattern alongside the counterstain; chromogenic granules alone do not resolve mitochondrial subcompartments (standard IHC interpretation).
Predominantly nuclear, membranous or extracellular signal.	This is a compartment mismatch for the principal mitochondrial pattern (HPA subcellular; UniProt Q12931). HPA also lists nucleoplasm as an additional ICC-IF location, so nuclear signal alone cannot prove an artefact; review its strength, distribution and controls (HPA subcellular; standard IHC interpretation).
Strong colour in cells outside the expected population.	First check the cell identification: HPA describes broad tissue expression, so an unlisted cell type is not automatically negative (HPA tissue IHC). A discordant pattern may reflect cross-reactivity or endogenous detection activity; inspect controls before assigning it to TRAP1 (standard IHC interpretation).
Diffuse colour across cells, stroma or the whole section.	A uniform haze obscures the reported granular cytoplasmic pattern (HPA tissue IHC). Review background in the no-primary control, washing and detection conditions; these checks address nonspecific staining and do not establish a TRAP1-specific cause (standard IHC practice).
No signal in a tissue expected to stain strongly.	Absent staining in adrenal glandular cells or bronchial respiratory epithelial cells conflicts with their reported High levels (HPA tissue IHC). Check tissue preservation and the assay controls before interpreting a test specimen as TRAP1-negative (standard IHC practice).

💡Expected TRAP1 appearanceCall a positive result when expected cells show convincing granular cytoplasmic staining, potentially strong in HPA High populations; isolated nuclear colour or diffuse haze is insufficient (HPA tissue IHC; HPA subcellular; standard IHC interpretation).

How each factor affects the staining
Cellular localisation	TRAP1 is assigned to mitochondria, including matrix and inner membrane, without a transmembrane segment (UniProt Q12931 topology). Paraffin-section chromogenic IHC should be read at the granular cytoplasmic level; it cannot distinguish matrix from inner membrane by appearance alone (standard IHC interpretation).
Tissue and cell context	HPA describes granular cytoplasmic expression across tissues and low tissue specificity at the RNA level (HPA tissue IHC). Its High examples identify useful positive cell populations, while the supplied record gives no validated negative tissue or expected intensity for every cell type (HPA tissue IHC).
Protein forms	UniProt lists two isoforms and a mature chain spanning residues 60–704 (UniProt Q12931). The supplied evidence does not map an IHC antibody epitope to either form, so these annotations cannot predict isoform-specific staining or an effect of processing on detection.
IF/ICC Q: What localisation should be seen?	A: Mainly mitochondrial signal is expected; HPA also records nucleoplasm as an additional location and rates the main mitochondrial location Enhanced (HPA subcellular). That ICC-IF observation supports a localisation cross-check, not an IHC protocol or proof that every nuclear IHC signal is specific.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
A reported High cell population has no chromogenic signal.	The run may have failed, or the sampled section may lack identifiable expected cells; the image alone cannot separate these possibilities (HPA tissue IHC; standard IHC interpretation).	Confirm the cells on the counterstain, then assess a known-positive section and the assay controls from the same run (standard IHC practice). Do not score the specimen negative until the positive control stains.
Staining appears mainly nuclear.	Nucleoplasm is an additional ICC-IF location, but the principal HPA localisation is mitochondrial (HPA subcellular). The IHC result may therefore be discordant or partly nonspecific; the supplied evidence cannot decide which.	Compare cytoplasmic granularity and nuclear colour in the same cells; check a no-primary control and, if available, an independently validated IHC antibody (HPA antibodies; standard IHC practice).
Stroma or unexpected structures are strongly coloured.	The distribution may reflect nonspecific binding or endogenous detection activity rather than the reported granular cytoplasmic cell pattern (HPA tissue IHC; standard IHC interpretation).	Inspect a no-primary control and the detection blank, then review blocking, washing and detection chemistry as general IHC controls (standard IHC practice). Avoid treating an unlisted cell type alone as proof of cross-reactivity (HPA tissue IHC).
The whole section has diffuse brown background.	General staining background can conceal cell-level granularity; HPA reports a granular cytoplasmic profile, not a uniform wash of colour (HPA tissue IHC).	Check the no-primary control and counterstain, then review wash steps and detection conditions (standard IHC practice). Interpret tissue intensity only after the expected cellular pattern is distinguishable.
Only a few cells stain within a broadly positive tissue.	HPA reports expression across tissues but identifies particular High cell populations; its summary does not require every cell in a section to stain equally (HPA tissue IHC).	Identify the stained and unstained cell populations morphologically, then compare the relevant HPA cell-level pattern and the run's positive control (HPA tissue IHC; standard IHC interpretation).
A putative positive looks smooth rather than granular.	The appearance does not match HPA's granular cytoplasmic IHC profile, although section resolution and chromogenic signal strength can limit visual detail (HPA tissue IHC; standard IHC interpretation).	Review the cells at suitable magnification and compare with a known-positive control; use the combined compartment, cell population and control results for the call (HPA tissue IHC; standard IHC practice).

Source: UniProt Q12931 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing TRAP1 staining

IHC analysis of TRAP1 using anti-TRAP1 antibody (PB9834). TRAP1 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-TRAP1 Antibody (PB9834) 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.

PB9834 · IHCIHC analysis of TRAP1 using anti-TRAP1 antibody (PB9834). TRAP1 was detected in a paraffin-embedded section of human intestinal cancer tissu

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000126602-TRAP1/tissue/cerebral+cortex#imid_15160154

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000126602-TRAP1/tissue/colon#imid_15160080

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000126602-TRAP1/tissue/liver#imid_15160081

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000126602-TRAP1/tissue/kidney#imid_15160082

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA041082; green = TRAP1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for TRAP1 IHC & IF

🧪Run appendix first and look for staining in glandular cells (HPA: High in appendix glandular cells). HPA detects TRAP1 in all 44 scored tissues, so use no-primary and isotype controls for the negative; cells without visible signal on the positive slide should remain at background, but HPA establishes no reliably negative internal cell population (HPA: no negative tissue rows).

Positive control tissue: Adrenal gland (Glandular cells, HPA High)

Negative control tissue: None in HPA: TRAP1 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show TRAP1 in A-431, MCF-7, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, siRNA 2 (10x), U2OS, siRNA 2 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), NIH 3T3, with annotated localisation: Mitochondria (enhanced) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only slide, an isotype-matched nonimmune rabbit IgG control, and TRAP1 knockout material as a biological negative (PB9834 caption: rabbit primary; standard IHC practice). For appendix sections, quench endogenous peroxidase and check for endogenous biotin background when using the caption’s biotin-based DAB detection (PB9834 caption: biotinylated secondary, SABC and DAB; standard IHC practice).

⚠️Feasibility: No target-specific fixation window or fixation effect is reported, and the selected paraffin-section caption does not state a fixative (PB9834 caption). Heat-mediated retrieval in EDTA at pH 8.0 is a documented IHC starting condition, though the caption does not establish that retrieval is required (PB9834 caption). Frozen-section feasibility and whether IF is easier are unreported; HPA’s cell-based ICC-IF images show predominantly mitochondrial localization, while appendix peroxidase or biotin background needs control in chromogenic IHC (HPA subcellular; standard IHC practice).

HPA tissue IHC evidence for TRAP1

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Enhanced — High 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
Adrenal gland	Glandular cells	High	Protein (IHC)	HPA →
Appendix	Glandular cells	High	Protein (IHC)	HPA →
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Breast	Adipocytes	High	Protein (IHC)	HPA →
Bronchus	Respiratory epithelial cells	High	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
None in HPA: TRAP1 is detected in all 44 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced TRAP1 IHC Tips

Troubleshoot TRAP1 staining in paraffin sections by checking retrieval, cellular pattern, controls, and scoring before interpreting changes in expression.

What should I try when TRAP1 staining is weak in paraffin sections?

Start with heat-mediated retrieval in EDTA at pH 8.0 (datasheet PB9834). The selected paraffin-section example used this retrieval before overnight incubation at 4°C with 1 μg/ml antibody; its fixative was not reported (caption PB9834). If staining remains weak, compare a small retrieval time series on matched sections while holding antibody concentration and detection constant, and inspect tissue integrity after heating (standard IHC practice). Judge improvement by a reproducible granular cytoplasmic pattern in appropriate cells, rather than by stronger staining at section edges or in damaged areas (HPA tissue IHC; standard IHC practice).

Could fixation explain variable TRAP1 staining between specimens?

Target-specific sensitivity of TRAP1 staining to fixative type or fixation duration is unknown from the supplied evidence; the selected paraffin-section caption does not report its fixative (caption PB9834). Record each specimen’s fixation and processing history, then compare matched sections using the same EDTA pH 8.0 retrieval and detection conditions (datasheet PB9834; standard IHC practice). Include a consistently processed reference section in each run to distinguish specimen variation from run variation (standard IHC practice). If signal differs across fixation groups, confirm the pattern with controls before attributing it to TRAP1 abundance or to a particular fixation effect (standard IHC practice).

What staining pattern is expected for TRAP1 in chromogenic IHC?

Look for granular cytoplasmic staining consistent with mitochondrial TRAP1; tissue IHC reports this pattern broadly, while subcellular evidence places TRAP1 mainly in mitochondria (HPA tissue IHC; HPA subcellular; UniProt Q12931 localisation). Mitochondrial matrix and inner-membrane annotations support an intracellular pattern, but chromogenic sections cannot resolve which side of the inner membrane contains the stained epitope (UniProt Q12931 localisation; standard IHC practice). Compare cells within the same section at similar thickness and development, since apparent granularity changes with focus and signal intensity (standard IHC practice). Treat prominent diffuse extracellular or uniform nuclear staining as a reason to check controls and morphology before calling it TRAP1 (HPA tissue IHC; standard IHC practice).

Can this IHC stain distinguish TRAP1 isoforms or modified epitopes?

Do not assign staining to a particular isoform without an antibody epitope map and isoform-specific validation: TRAP1 has 2 annotated isoforms, but the supplied product caption gives no such map (UniProt Q12931 isoforms; caption PB9834). The annotated mitochondrial chain spans residues 60–704, and several phosphorylation and acetylation sites are recorded, including residues 170 and 262 (UniProt Q12931 processing and modified residues). Those annotations alone do not establish whether processing or modification changes antibody binding in paraffin sections (standard IHC interpretation). If a biological comparison hinges on an isoform or modification, use separately validated, discriminating evidence alongside the chromogenic stain (standard IHC practice).

How can IF help check a puzzling TRAP1 IHC pattern?

Use IF/ICC as a separate localisation check: subcellular imaging reports TRAP1 mainly in mitochondria, with nucleoplasm as an additional location (HPA subcellular). Multiplex TRAP1 with a validated marker for the expected cell type, and choose spectrally separated fluorophores after checking the specimen’s autofluorescence with an unstained control (HPA tissue IHC; standard IF practice). Because TRAP1 is annotated in the mitochondrial matrix and inner membrane and has no transmembrane segment, optimise permeabilisation for access to an intracellular epitope; the supplied evidence does not identify the antibody’s epitope or specify an IF permeabilisation condition (UniProt Q12931 topology and localisation; caption PB9834). Keep IF localisation findings distinct from the paraffin-section chromogenic readout when resolving disagreement (standard IHC/IF practice).

How do I reduce widespread brown staining without losing TRAP1 signal?

First compare a no-primary control with the stained section, and check whether pigment, endogenous enzyme activity, or detection reagents contribute to the brown signal (standard chromogenic IHC practice). The selected example used 10% goat serum blocking, a biotinylated secondary, a streptavidin–biotin detection complex, and DAB; these describe that example, not a requirement for every specimen (caption PB9834). Where needed, apply a peroxidase block and evaluate endogenous biotin interference with the detection system in use (standard chromogenic IHC practice). Titrate antibody and development while preserving granular cytoplasmic staining, and reject improvements that merely suppress both background and the reference section’s signal (HPA tissue IHC; standard IHC practice).

How should I score TRAP1 across paraffin-section samples? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and compartment before scoring, using granular cytoplasmic staining as the expected tissue pattern (HPA tissue IHC). For a cell-based comparison, record the percentage of positive cells and staining intensity, then calculate an H-score using the same thresholds across sections; alternatively, report positive-cell density per mm² of viable tissue (standard IHC practice). Normalise to the number or area of the relevant cells and exclude necrosis, folds, and section edges from the analysis region (standard IHC practice). Keep retrieval, antibody incubation, DAB development, imaging, and threshold settings consistent, and include a reference section to detect run variation (standard IHC practice).

When is a change in TRAP1 staining biologically credible?

A credible signal follows the expected granular cytoplasmic pattern in morphologically intact cells and is reproducible with appropriate controls (HPA tissue IHC; standard IHC practice). The selected paraffin-section image used human intestinal cancer tissue, but that example alone does not establish a universal cancer-associated increase (caption PB9834; standard IHC interpretation). UniProt reports reduced expression in bladder cancer and renal cell carcinoma specimens and increased expression in other tumor types, so interpret changes within the sampled tissue and cell population (UniProt Q12931 tissue specificity). Recheck dominant extracellular or diffuse nuclear staining, section-edge enhancement, necrotic areas, and residual endogenous enzyme signal before treating a brown deposit as a biological difference (HPA tissue IHC; standard IHC practice).

Boster reagents

Best TRAP1 / Heat shock protein 75 kDa, mitochondrial IHC Antibodies

PB9834 has a human paraffin-section IHC figure (PB9834 IHC caption). M02426-4 lists human IHC and IF/ICC applications, but has no supplied figure captions (catalog applications; catalog image alts).

Real IHC data

Anti-TRAP1 Antibody ®

Cat # PB9834

View product →

PB9834 lists human and rat reactivity and has an IHC figure from a human intestinal cancer paraffin section (catalog reactivity; PB9834 IHC caption). M02426-4 lists human reactivity and IHC and IF/ICC applications, with no supplied IHC or IF figure captions (catalog reactivity/applications; catalog image alts).

Which to pick: For tissue IHC, choose PB9834: its paraffin-section figure used EDTA retrieval at pH 8.0 and 1 μg/ml primary antibody; the fixative is unreported (PB9834 IHC caption). For IF/ICC, M02426-4 is a rabbit monoclonal, clone 23T98, listed for human IF/ICC at 1:50, though no IF figure caption is supplied (catalog clone/reactivity/applications/dilution; catalog IF image alts). For human and rat reactivity, PB9834 lists both species, while its supplied IHC figure documents human tissue only (catalog reactivity; PB9834 IHC caption).

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

References

UniProt Consortium. UniProt entry Q12931 (TRAP1_HUMAN, Heat shock protein 75 kDa, mitochondrial).
Human Protein Atlas. TRAP1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. TRAP1 subcellular location (ICC-IF): Mainly localized to the mitochondria..
Human Protein Atlas. TRAP1 antibody validation summary (2 antibodies).
Role of TRAP1 and estrogen receptor alpha in patients with ovarian cancer -a study of the OVCAD consortium. Molecular cancer 2012 — PMC3533746.
TRAP1 protein signature predicts outcome in human metastatic colorectal carcinoma. Oncotarget 2017 — PMC5400579.
TRAP1 regulates proliferation, mitochondrial function, and has prognostic significance in NSCLC. Molecular cancer research : MCR 2014 — PMC5207305.
Aberrantly upregulated TRAP1 is required for tumorigenesis of breast cancer. Oncotarget 2015 — PMC4792571.
PubMed PMID:10545594 — UniProt-cited evidence.
PubMed PMID:14702039 — UniProt-cited evidence.
PubMed PMID:15616553 — UniProt-cited evidence.