SPAG5 / Sperm-associated antigen 5 · IHC design guide

Design Immunohistochemistry for SPAG5

Plan SPAG5 paraffin IHC with the catalog antibody at 2–5 µg/mL (datasheet A07062-2). Compare cytoplasmic staining in testis and kidney tubules with a negative-control section, while accounting for low consistency between antibody staining and RNA expression (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for SPAG5 (IHC for SPAG5): expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody A07062-2, validated IHC image, and IHC protocol steps — Printable **SPAG5** IHC protocol sheet — expected localisation Cytoplasmic staining in most tissues (HPA tissue IHC), antibody A07062-2, controls and protocol steps. Open the full SPAG5 IHC guide →

Navigate to: Protocols Staining pattern Controls Antibodies

SPAG5 Immunohistochemistry Experimental Design Guide

Expected localisation, validated protocols, controls and antibodies — the at-a-glance facts below, then the full design guide.

Must know before staining

	Expected localisation	Cytoplasmic staining in most tissues (HPA tissue IHC)
	Staining pattern	Variable cytoplasmic staining across most tissues, including testis (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A07062-2)
	Positive control ⓘ	Bone marrow+4 more · see all
	Negative control ⓘ	Heart muscle+4 more · see all

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep paraffin-section fixation consistent across samples. (standard IHC practice; not target-specific)
	Caveat	Antibody staining has low consistency with RNA expression (HPA tissue IHC)
	Regulation	Tissue enhanced RNA: marrow, kidney, testis (HPA tissue IHC: RNA specificity)
	Isoform / epitope	No annotated isoforms or processing variants (UniProt)

Section 1

Recommended SPAG5 IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet: A07062-2) is accompanied by three published SPAG5 IHC protocols (PMC9116224; PMC11657553; PMC7007403).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded human renal clear cell carcinoma tissue; fixative not specified (datasheet A07062-2)
Fixation	Image fixative and duration unreported (datasheet A07062-2); 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 A07062-2); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A07062-2)
Primary antibody	Rabbit anti-SPAG5, 2-5μg/ml (datasheet A07062-2)
Primary incubation	Overnight at 4 °C (datasheet A07062-2)
Detection	Streptavidin-biotin complex (SABC), DAB chromogen (datasheet A07062-2)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	SPAG5-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Cytoplasmic expression at variable levels in most tissues, including testis. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0 (datasheet: A07062-2); sodium citrate at 95–98°C for 10 min is a published alternative (PMC9116224).

Section 2

What Is the Expected SPAG5 Staining Pattern?

SPAG5 is a non-membrane protein associated with cytoplasm, microtubules and the mitotic spindle (UniProt Q96R06 topology and subcellular location). In paraffin sections, expect cytoplasmic staining in cell populations reported positive by HPA, particularly seminiferous duct cells, kidney tubule cells and bone marrow hematopoietic cells (HPA tissue IHC: High). Interpret intensity cautiously: HPA rates its tissue IHC staining Approved but reports low consistency with RNA expression (HPA tissue IHC reliability).

What am I looking at on my slide?
Distinct cytoplasmic staining in seminiferous duct cells, kidney tubules or bone marrow hematopoietic cells.	This fits the reported High staining in those cell populations (HPA tissue IHC). Compare the named cells with adjacent cells; HPA's levels describe observed staining, not an intensity threshold for every cell (HPA tissue IHC).
Signal concentrated around mitotic figures, with less conspicuous staining in neighboring cells.	A spindle-associated pattern is biologically plausible: SPAG5 occurs at spindle poles, across the spindle and later at midzone microtubules as mitosis progresses (UniProt Q96R06 subcellular location). A chromogenic section may not resolve each structure (general IHC practice).
Predominantly nuclear-only staining across many cells, without a convincing cytoplasmic pattern.	Reassess compartment assignment, counterstain and detection controls before calling it SPAG5 (general IHC practice). HPA describes cytoplasmic tissue IHC, although its separate ICC-IF data support nuclear bodies; nuclear signal alone is therefore not a universal artefact (HPA tissue IHC; HPA subcellular ICC-IF).
Strong staining in cardiomyocytes or pancreatic exocrine glandular cells.	Those specific cell populations were not detected in HPA tissue IHC. Check morphology and controls for possible cross-reactivity or endogenous detection activity before treating the signal as SPAG5 (HPA tissue IHC; general IHC practice).
Diffuse deposit obscures cell boundaries, or no signal appears in a known-positive comparison section.	Diffuse background prevents a reliable compartment call; a blank positive comparison leaves assay performance unresolved (general IHC practice). Neither result alone establishes SPAG5 absence in the test section, especially given the reported variability in tissue staining (HPA tissue IHC).

💡Expected SPAG5 appearanceCall a positive IHC result when discernible cytoplasmic staining occurs in the specified HPA-positive cell populations, potentially highlighting mitotic structures; widespread featureless deposit or strong signal in HPA-negative cell types warrants review (HPA tissue IHC; UniProt Q96R06 subcellular location).

How each factor affects the staining
Cell population and tissue	HPA reports High staining in bone marrow hematopoietic cells, kidney tubules and testis seminiferous ducts, but not detected in cardiomyocytes (HPA tissue IHC). Score the identified cells, not the entire section.
Cell-cycle state	Interphase SPAG5 can be punctate; mitotic localization shifts from spindle poles to spindle and midzone structures (UniProt Q96R06 subcellular location). The visible pattern can therefore vary among cells in one section.
Strength of tissue evidence	HPA labels tissue IHC Approved while noting low staining–RNA consistency; two listed antibodies have IHC Approved status (HPA tissue IHC; HPA antibodies). Treat an unexpected pattern as a finding to verify.
Topology and processing	UniProt lists no transmembrane segment or signal peptide and annotates one full-length chain (UniProt Q96R06 topology and processing). These annotations support an intracellular expectation; they do not predict retrieval or fixation sensitivity.
IF/ICC Q: Should its pattern match tissue IHC?	HPA ICC-IF supports nuclear bodies and mitotic spindle, with additional cytosol, whereas tissue IHC describes variable cytoplasmic staining (HPA subcellular ICC-IF; HPA tissue IHC). Interpret each application with its own evidence.

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
No staining in the test section or the positive comparison.	Assay failure remains possible; HPA reports High staining in the named testis, kidney and marrow cell populations (HPA tissue IHC).	Repeat with a documented positive tissue and verify the IHC-P retrieval, antibody and chromogenic detection steps used in the run (general IHC practice).
The positive comparison stains, but the test section does not.	Expression may differ by cell population; HPA also reports several specific cell types as not detected (HPA tissue IHC).	Identify the cell type on the counterstain and compare its reported HPA level before interpreting the absence (HPA tissue IHC; general IHC practice).
A HPA-negative cell population stains strongly.	Cross-reactivity or endogenous detection activity is possible; the discrepancy alone cannot identify its cause (HPA tissue IHC; general IHC practice).	Inspect antibody-omission and detection controls, then reassess morphology and the affected cell population (general IHC practice).
Diffuse chromogen obscures intracellular detail.	Background prevents a dependable cytoplasmic or spindle-associated interpretation (general IHC practice).	Review blocking, antibody dilution, washing and detection controls using the established IHC-P workflow; reassess only after cell boundaries are readable (general IHC practice).
Signal appears nuclear-only in tissue IHC.	It differs from HPA's cytoplasmic tissue description, although nuclear bodies are supported in ICC-IF (HPA tissue IHC; HPA subcellular ICC-IF).	Check counterstain alignment and controls, then report the application and compartment explicitly instead of equating the two HPA patterns (general IHC practice; HPA subcellular ICC-IF).
A positive section shows uneven intensity among cells.	HPA describes variable cytoplasmic tissue staining, and UniProt describes cell-cycle-dependent localization (HPA tissue IHC; UniProt Q96R06 subcellular location).	Score defined cell populations and compartments separately; compare sections using the same detection and scoring conditions (general IHC practice).

Source: UniProt Q96R06 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing SPAG5 staining

IHC analysis of Astrin/Deepest/SPAG5 using anti-Astrin/Deepest/SPAG5 antibody (A07062-2). Astrin/Deepest/SPAG5 was detected in paraffin-embedded section of human renal clear cell carcinoma 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-Astrin/Deepest/SPAG5 Antibody (A07062-2) 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.

A07062-2 · IHCIHC analysis of Astrin/Deepest/SPAG5 using anti-Astrin/Deepest/SPAG5 antibody (A07062-2). Astrin/Deepest/SPAG5 was detected in paraffin-embe

IF analysis of Astrin/Deepest/SPAG5 using anti-Astrin/Deepest/SPAG5 antibody (A07062-2). Astrin/Deepest/SPAG5 was detected in immunocytochemical section of U20S 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 5μg/mL rabbit anti-Astrin/Deepest/SPAG5 Antibody (A07062-2) 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.

A07062-2 · IFIF analysis of Astrin/Deepest/SPAG5 using anti-Astrin/Deepest/SPAG5 antibody (A07062-2). Astrin/Deepest/SPAG5 was detected in immunocytochem

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000076382-SPAG5/tissue/cerebral+cortex#imid_6119064

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000076382-SPAG5/tissue/colon#imid_6119006

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000076382-SPAG5/tissue/liver#imid_6118999

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000076382-SPAG5/tissue/kidney#imid_6118992

HPA · ICC-IF HAP1Human Protein Atlas immunofluorescence, HAP1, antibody HPA024418; green = SPAG5, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for SPAG5 IHC & IF

🧪Run testis first and expect staining in cells of the seminiferous ducts (HPA: High in cells in seminiferous ducts; UniProt: highly expressed in testis). Run heart muscle as the negative tissue and expect cardiomyocytes to remain unstained (HPA: Not detected in cardiomyocytes); on the positive slide, cells without specific staining should show only background and can serve as internal comparisons (HPA: High in cells in seminiferous ducts).

Positive control tissue: Bone marrow (Hematopoietic cells, HPA High)

Negative control tissue: Heart muscle (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show SPAG5 in HAP1, Rh30, U2OS, with annotated localisation: Nuclear bodies (supported), Mitotic spindle (supported) (HPA subcellular).

Technical controls: Include a no-primary, secondary-only control and a concentration-matched nonimmune rabbit IgG isotype control, matched to the primary’s clonality if known (selected-SKU caption: rabbit primary and goat anti-rabbit secondary); use a SPAG5-knockout specimen or peptide-blocked primary as a biological specificity control if available (standard IHC practice). Block endogenous peroxidase and check for endogenous biotin in testis when using the caption’s SABC–DAB detection system (selected-SKU caption: SABC–DAB).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact selected-SKU paraffin-section caption does not state a fixative (selected-SKU caption: fixative not stated). The caption uses heat-mediated EDTA retrieval at pH 8.0, but whether retrieval is required is unreported; the supplied evidence also does not establish whether frozen sections or IF are easier (selected-SKU caption: EDTA retrieval; HPA: ICC-IF localisation data). For testis IHC, assess endogenous biotin when using SABC detection because it can produce background staining (selected-SKU caption: SABC detection; standard IHC practice).

HPA tissue IHC evidence for SPAG5

Comprehensive Human Protein Atlas IHC scoring per tissue (reliability: Approved — Low 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
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Kidney	Cells in tubules	High	Protein (IHC)	HPA →
Testis	Cells in seminiferous ducts	High	Protein (IHC)	HPA →
Tonsil	Non-germinal center cells	High	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Heart muscle	Cardiomyocytes	Not detected	Protein (IHC)	HPA →
Oral mucosa	Squamous epithelial cells	Not detected	Protein (IHC)	HPA →
Pancreas	Exocrine glandular cells	Not detected	Protein (IHC)	HPA →
Salivary gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Skeletal muscle	Myocytes	Not detected	Protein (IHC)	HPA →

Section 3

Advanced SPAG5 IHC Tips

Use cell type, compartment and staining controls to assess SPAG5 in paraffin section IHC (HPA tissue IHC; UniProt Q96R06).

What retrieval should I try first if SPAG5 staining is weak?

Start with heat mediated antigen retrieval in EDTA at pH 8.0 (datasheet A07062-2). The selected paraffin section image used this retrieval before 2 µg/mL primary antibody overnight at 4°C, so keep those conditions together for an initial comparison (datasheet A07062-2). If staining remains weak, adjust retrieval heating and cooling in a small matched section series while holding detection and exposure conditions constant (standard IHC practice). Compare signal in the expected cells with a no primary control, because stronger overall DAB color alone does not establish improved SPAG5 detection (HPA tissue IHC; standard IHC practice).

How should I troubleshoot fixation related loss of SPAG5 staining?

The selected paraffin section caption does not state a fixative, so SPAG5 sensitivity to fixation is unknown for this antibody (datasheet A07062-2). Record each specimen's fixative and fixation duration, then compare sections processed with the same retrieval, antibody concentration and detection conditions (standard IHC practice). Use the reported EDTA pH 8.0 retrieval and 2 µg/mL antibody as a reproducible starting point, without treating them as evidence for any fixation condition (datasheet A07062-2). If a fixation difference tracks signal loss, assess matched positive tissue and no primary controls before changing retrieval, since processing and background can also affect chromogenic contrast (HPA tissue IHC; standard IHC practice).

Which staining compartments are plausible for SPAG5 in tissue sections?

Assess cytoplasmic staining in the context of cell identity and mitotic activity: tissue IHC reports variable cytoplasmic expression, including testis (HPA tissue IHC). SPAG5 can occupy spindle poles, the mitotic spindle, kinetochores and the midbody as cells progress through mitosis (UniProt Q96R06 localisation). Interphase puncta and centrosomal or centriolar satellite association are also described, although a chromogenic section may not resolve each structure (UniProt Q96R06 localisation; standard IHC practice). Review suspicious nuclear staining against morphology and controls; nuclear bodies are supported by subcellular IF evidence, but diffuse nuclear DAB should not automatically be scored as SPAG5 (HPA subcellular; standard IHC practice).

Can isoforms or modifications explain discordant SPAG5 staining?

The supplied record lists one SPAG5 chain spanning residues 1–1193 and zero annotated isoforms; it does not establish the catalog antibody's epitope (UniProt Q96R06 record). SPAG5 has multiple annotated phosphorylation sites, but their effect on this antibody's binding in paraffin sections is unknown (UniProt Q96R06 modified residues; datasheet A07062-2). When two antibodies disagree, compare their stated immunogens or epitopes if available, along with matched retrieval and detection controls (standard IHC practice). Do not assign a staining difference to an isoform or phosphorylation event without epitope information and independent validation, such as concordant staining with an antibody recognizing another region (standard IHC practice).

How can IF help assess a disputed SPAG5 IHC pattern?

Use IF as an orthogonal localisation check alongside chromogenic IHC, since supported IF locations include the mitotic spindle, nuclear bodies and cytosol (HPA subcellular). Multiplex SPAG5 with a validated marker defining the expected cell type, then compare its signal with morphology and the matching IHC section (HPA tissue IHC; standard IF practice). Choose a fluorophore in a channel with low tissue autofluorescence, often a longer wavelength channel, and include single stain and no primary controls (standard IF practice). SPAG5 has no annotated transmembrane segment and occupies intracellular structures, so permeabilise sufficiently for intracellular epitope access while checking that spindle and punctate patterns remain intact (UniProt Q96R06 topology and localisation; standard IF practice).

How do I reduce diffuse DAB or focal false positive staining?

Begin with a no primary control and inspect whether deposits follow tissue edges, damaged areas or pigment rather than cellular compartments (standard IHC practice). The selected image used 10% goat serum blocking, a biotinylated secondary antibody and a streptavidin biotin detection system with DAB; those details define the comparison workflow (datasheet A07062-2). Include a peroxidase block and consider endogenous biotin when evaluating signal from this detection chemistry (standard IHC practice; datasheet A07062-2). If background persists, titrate the primary around the reported 2 µg/mL condition and adjust washing or DAB development using matched sections, while checking that expected cellular staining remains (datasheet A07062-2; standard IHC practice).

What scoring method best captures SPAG5 IHC heterogeneity? ⚠ ANSWER MARKED FOR VERIFICATION

Define the scored cell population and compartment before reviewing slides, because SPAG5 staining can vary across tissues and mitotic states (HPA tissue IHC; UniProt Q96R06 localisation). For broad cytoplasmic staining, report the percentage of positive target cells and an H-score calculated from intensity categories; use identical thresholds across the cohort (standard IHC practice). For discrete mitotic figures, report SPAG5 positive cells per mm² or per counted target cells, and record the mitotic fraction separately (standard IHC practice; UniProt Q96R06 function). Normalize counts to viable tissue area or the relevant cell population, excluding necrosis and section edges consistently, and retain staining controls for each batch (standard IHC practice).

When is a strong SPAG5 IHC signal convincing?

A convincing result follows cell morphology and plausible compartments: tissue IHC reports high staining in kidney tubule cells, bone marrow hematopoietic cells and testis seminiferous duct cells (HPA tissue IHC). Mitotic spindle associated staining is biologically plausible, while a strong diffuse signal confined to section edges or necrotic areas warrants scrutiny (UniProt Q96R06 localisation; standard IHC practice). Check no primary and detection controls for endogenous enzyme or detection system signal before calling DAB deposits positive (standard IHC practice). Interpret weak or absent staining cautiously: HPA labels the tissue IHC data approved but notes low consistency with RNA expression, so staining alone should not establish expression differences (HPA tissue IHC).

Boster reagents

Best SPAG5 / Sperm-associated antigen 5 IHC Antibodies

A07062-2 has human paraffin-section IHC images and a U20S-cell IF image (catalog images); its listed reactivity is human (catalog: reactivity).

Real IHC data

Anti-Astrin/Deepest/SPAG5 Antibody ®

Cat # A07062-2

View product →

A07062-2 has IHC images from human renal clear cell carcinoma, liver cancer, lung cancer, and rectal cancer paraffin sections (A07062-2 IHC captions). It also has an IF image from U20S cells (A07062-2 IF caption).

Which to pick: Choose A07062-2 for human paraffin-section IHC: its renal clear cell carcinoma image documents EDTA retrieval at pH 8.0 and 2 μg/ml primary antibody (A07062-2 IHC caption); the fixative is unreported (A07062-2 IHC captions). The same SKU lists IF/ICC and shows IF in U20S cells at 5 μg/ml (A07062-2 applications and IF caption). No cross-species choice is supported because the listed reactivity is human only; clonality is unreported (A07062-2 catalog).

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

References

UniProt Consortium. UniProt entry Q96R06 (SPAG5_HUMAN, Sperm-associated antigen 5).
Human Protein Atlas. SPAG5 tissue IHC expression (reliability: Approved).
Human Protein Atlas. SPAG5 subcellular location (ICC-IF): Mainly localized to the nuclear bodies and mitotic spindle. In addition localized to the cytosol..
Human Protein Atlas. SPAG5 antibody validation summary (3 antibodies).
Comprehensive assessment of cellular senescence in the tumor microenvironment. Briefings in bioinformatics 2022 — PMC9116224.
SPAG5 is a potential therapeutic target affecting proliferation, apoptosis, and invasion of esophageal cancer cells. European journal of medical research 2024 — PMC11657553.
miR-539 inhibits prostate cancer progression by directly targeting SPAG5. Journal of experimental & clinical cancer research : CR 2016 — PMC4818461.
High expression of sperm-associated antigen 5 correlates with poor survival in ovarian cancer. Bioscience reports 2020 — PMC7007403.
PubMed PMID:11724960 — UniProt-cited evidence.
PubMed PMID:11549262 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.