BCLAF1 / Bcl-2-associated transcription factor 1 · IHC design guide

Design Immunohistochemistry for BCLAF1

Plan chromogenic BCLAF1 IHC in paraffin sections around its widespread nuclear staining (HPA tissue IHC). This guide covers fixation consistency, antibody selection and scoring, with high staining in bone marrow hematopoietic cells as a reference (HPA tissue IHC).

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

Immunohistochemistry protocol sheet for BCLAF1 (IHC for BCLAF1): expected localisation Nuclear staining across tissues (HPA tissue IHC), antibody A04100-3, validated IHC image, and IHC protocol steps — Printable **BCLAF1** IHC protocol sheet — expected localisation Nuclear staining across tissues (HPA tissue IHC), antibody A04100-3, controls and protocol steps. Open the full BCLAF1 IHC guide →

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BCLAF1 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	Nuclear staining across tissues (HPA tissue IHC)
	Staining pattern	Nuclear staining in glandular and hematopoietic cells (HPA tissue IHC)
	Antigen retrieval	EDTA pH 8.0 HIER, heat-mediated (datasheet A04100-3)
	Positive control ⓘ	Adrenal gland+4 more · see all
	Negative control ⓘ	None in HPA (detected in all 45 tissues); use no-primary + isotype controls

Important caveats

Reasons your staining may differ from the expected pattern.

	Fixation	Keep fixation conditions consistent across sections. (standard IHC practice; not target-specific)
	Caveat	Broad nuclear expression limits negative tissue controls (HPA tissue IHC)
	Regulation	Ubiquitous expression (UniProt)
	Isoform / epitope	4 isoforms; antibody epitope coverage is unknown (UniProt)

Section 1

Recommended BCLAF1 IHC & IF Protocols

The catalog antibody’s IHC-P protocol is followed by published BCLAF1 staining methods for tissue sections and tumor samples (datasheet A04100-3; PMC11844276; PMC11496133; PMC8486183).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded mouse brain tissue; fixative not specified (datasheet A04100-3)
Fixation	Image fixative and duration unreported (datasheet A04100-3); 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 A04100-3); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% goat serum (datasheet A04100-3)
Primary antibody	Rabbit anti-BCLAF1, 2-5 μg/ml (datasheet A04100-3)
Primary incubation	Overnight at 4 °C (datasheet A04100-3)
Detection	HRP-conjugated secondary, DAB chromogen (datasheet A04100-3)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	BCLAF1-positive staining in glandular cells of adrenal gland (HPA tissue IHC: High). HPA tissue profile: Ubiquitous nuclear expression. No signal in the no-primary control.

💡Decision noteStart with heat-mediated EDTA retrieval at pH 8.0: it matches the catalog datasheet and a published BCLAF1 IHC method (datasheet A04100-3; PMC8486183).

Section 2

What Is the Expected BCLAF1 Staining Pattern?

In paraffin sections, expect BCLAF1 chiefly in nuclei across many cell types, including glandular, hematopoietic, respiratory epithelial and glial cells (HPA tissue IHC: ubiquitous nuclear expression; Enhanced reliability). Nuclear speckles and nucleoplasm are annotated locations; cytoplasm is also listed, so a small cytoplasmic component cannot be excluded (UniProt Q9NYF8). BCLAF1 has no transmembrane segment (UniProt Q9NYF8 topology).

What am I looking at on my slide?
Nuclear chromogen in several cell types, with strong staining in bone marrow hematopoietic cells.	This fits the reported ubiquitous nuclear pattern and a documented High tissue example (HPA tissue IHC). Compare nuclei within the same section; the HPA examples do not establish one required intensity for every cell.
Predominantly cytoplasmic chromogen with little or no nuclear staining.	This conflicts with the tissue IHC pattern and warrants an artefact check (HPA tissue IHC: ubiquitous nuclear expression). Cytoplasm is also a UniProt location, so cytoplasmic signal alone does not prove nonspecific binding (UniProt Q9NYF8).
Chromogen concentrated in anuclear material or structures while expected nuclei are unstained.	Treat the distribution as suspect for cross-reactivity or endogenous detection activity (general IHC practice). HPA reports nuclear staining across tissues, so staining of such material cannot establish BCLAF1 expression (HPA tissue IHC).
Diffuse colour obscures nuclei and tissue boundaries.	The slide cannot be scored confidently for the expected nuclear pattern (HPA tissue IHC). Excess detection background, incomplete blocking or inadequate washing are possible workflow causes; the appearance alone does not identify which one (general IHC practice).
No nuclear signal in bone marrow hematopoietic cells.	Investigate the run because these cells are reported High in HPA tissue IHC. A negative slide alone cannot distinguish a failed stain from sample variation or an antibody-specific issue; review controls and tissue preservation (general IHC practice).

💡Expected BCLAF1 appearanceCall a slide positive when interpretable nuclei show chromogen across represented cell types, potentially High in bone marrow hematopoietic cells; isolated staining of anuclear material is suspect (HPA tissue IHC; general IHC practice).

How each factor affects the staining
Tissue and cell distribution	HPA reports ubiquitous nuclear expression and High staining in the listed glandular, hematopoietic, respiratory epithelial and glial examples (HPA tissue IHC). Its supplied examples contain no designated negative tissue, so they cannot define a negative tissue control.
Antibody evidence for IHC	HPA006669 and HPA027770 have IHC Enhanced validation; HPA006484 has no IHC status in the supplied record (HPA antibodies). Enhanced supports confidence in the observed pattern but does not guarantee identical results with another antibody or specimen.
Subcellular interpretation	Nucleus, nucleoplasm, nuclear speckles and cytoplasm are annotated; no transmembrane segment is reported (UniProt Q9NYF8). Judge chromogenic paraffin sections against the HPA nuclear tissue pattern, without treating every cytoplasmic pixel as decisive.
Isoforms and epitope scope	UniProt lists 4 isoforms (UniProt Q9NYF8). The supplied record gives no antibody epitope or isoform coverage, so it cannot predict which isoforms contribute to an IHC signal or explain an absent stain.
Processing and modification	The annotated chain spans residues 1–920, with no signal peptide or propeptide; modified residues are reported (UniProt Q9NYF8). These annotations do not establish shedding, IHC epitope accessibility or fixation sensitivity.
IF/ICC Q: what pattern is expected?	A: HPA reports enhanced localization to nuclear speckles in ICC-IF (HPA subcellular). This supports a punctate nuclear expectation for that application; the chromogenic tissue IHC profile remains the reference for paraffin-section scoring (HPA tissue IHC).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Bone marrow shows no nuclear chromogen.	Possible run or specimen problem; HPA reports High hematopoietic-cell staining but gives no target-specific failure mechanism (HPA tissue IHC).	Check a concurrent positive tissue, counterstain and detection controls; verify the antibody's IHC validation and the documented workflow (general IHC practice; HPA antibodies).
Only cytoplasm appears positive.	The pattern differs from HPA's ubiquitous nuclear tissue profile, although UniProt also lists cytoplasm (HPA tissue IHC; UniProt Q9NYF8).	Review nuclear morphology, localization at higher magnification and a detection control before assigning the signal to BCLAF1 (general IHC practice).
Strong colour appears in anuclear material.	Cross-reactivity or endogenous detection activity may contribute; location alone cannot identify the cause (general IHC practice).	Inspect no-primary and detection controls, then assess whether staining also follows the expected nuclear distribution (general IHC practice; HPA tissue IHC).
Background hides nuclear boundaries.	Detection background, incomplete blocking or insufficient washing can obscure compartment scoring (general IHC practice).	Review controls and optimize blocking, washing and detection conditions one variable at a time; rescore only when nuclei are distinguishable (general IHC practice).
Staining varies between cell types.	HPA reports ubiquitous nuclear expression, with High levels in selected cell types; equal intensity in every cell is not established (HPA tissue IHC).	Score nuclear localization and intensity by identifiable cell type, and use the HPA examples as references rather than an all-cells-equal threshold (HPA tissue IHC).
IF speckles seem inconsistent with IHC staining.	HPA reports nuclear speckles for ICC-IF and a ubiquitous nuclear pattern for tissue IHC; the supplied evidence does not equate their visual resolution (HPA subcellular; HPA tissue IHC).	Interpret each image using its application-specific HPA pattern; do not require visible speckles to call a chromogenic paraffin-section nucleus positive (HPA subcellular; HPA tissue IHC).

Source: UniProt Q9NYF8 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing BCLAF1 staining

IHC analysis of BCLAF1 using anti-BCLAF1 antibody (A04100-3). BCLAF1 was detected in a paraffin-embedded section of mouse brain 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-BCLAF1 Antibody (A04100-3) 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.

A04100-3 · IHCIHC analysis of BCLAF1 using anti-BCLAF1 antibody (A04100-3). BCLAF1 was detected in a paraffin-embedded section of mouse brain tissue. Heat

IF analysis of BCLAF1 using anti-BCLAF1 antibody (A04100-3) and anti-Beta Tubulin antibody (M01857-3). BCLAF1 was detected in an immunocytochemical section of Hela 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-BCLAF1 Antibody (A04100-3) and mouse anti-Beta Tubulin antibody (M01857-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Rabbit IgG (BA1127) and Cy3 Conjugated Goat Anti-Mouse IgG (BA1031) were used as secondary antibody at 1:500 dilution and incubated for 30 minutes at 37°C. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

A04100-3 · IFIF analysis of BCLAF1 using anti-BCLAF1 antibody (A04100-3) and anti-Beta Tubulin antibody (M01857-3). BCLAF1 was detected in an immunocytoc

A04100-1 · IHCImmunohistochemistry of BCLAF1 in mouse brain tissue with BCLAF1 antibody at 5 μg/mL.

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000029363-BCLAF1/tissue/cerebral+cortex#imid_6791744

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000029363-BCLAF1/tissue/colon#imid_6791662

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000029363-BCLAF1/tissue/liver#imid_6791671

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000029363-BCLAF1/tissue/kidney#imid_6791672

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA027770; green = BCLAF1, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for BCLAF1 IHC & IF

🧪Start with breast: its glandular cells should show BCLAF1 staining (HPA: High in breast glandular cells). HPA detects BCLAF1 in all 45 scored tissues, so there is no supported negative tissue; no-primary and isotype sections carry the negative control, while cells without detectable signal on the positive slide should show counterstain without brown DAB and should not be assumed biologically BCLAF1-negative (HPA: detected in all 45 scored tissues; standard IHC practice).

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

Negative control tissue: None in HPA: BCLAF1 is detected in all 45 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 BCLAF1 in A-431, U-251MG, U2OS, Rh30, SiHa, with annotated localisation: Nuclear speckles (enhanced) (HPA subcellular).

Technical controls: Run a no-primary, secondary-only section and a concentration-matched rabbit IgG isotype section, plus BCLAF1-knockout material processed in parallel if available (caption: rabbit primary and HRP/DAB detection; standard IHC practice). Quench endogenous peroxidase and check the breast section for pigment or nonspecific DAB before scoring (caption: HRP/DAB detection; standard IHC practice).

⚠️Feasibility: A target-specific fixation window or fixation effect is unreported, and the exact A04100-3 paraffin-section caption does not state its fixative (caption: A04100-3). Heat retrieval in EDTA at pH 8.0 is a documented starting condition for this antibody; the caption used 2 μg/ml primary overnight at 4°C (caption: A04100-3). The supplied evidence does not establish that frozen sections or IF are easier; IF would need its own fixation and permeabilisation optimization, with nuclear speckles as a localisation check (HPA: ICC-IF nuclear speckles; standard IF practice).

HPA tissue IHC evidence for BCLAF1

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	Glandular cells	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: BCLAF1 is detected in all 45 scored tissues. Use a no-primary (secondary-only) and an isotype control instead.

Section 3

Advanced BCLAF1 IHC Tips

Troubleshoot BCLAF1 staining in paraffin sections by checking retrieval, nuclear localisation, controls, and scoring before interpreting biological differences.

Which retrieval condition should I try first for weak BCLAF1 staining?

Start with heat-mediated retrieval in EDTA at pH 8.0 for paraffin sections (datasheet A04100-3). The selected tissue image used this retrieval, followed by 10% goat serum blocking and 2 μg/ml primary antibody overnight at 4°C (datasheet A04100-3). If nuclear staining remains weak, compare retrieval heating and cooling conditions on adjacent sections while keeping antibody concentration and detection constant (standard IHC practice). Include a control section expected to show nuclear staining, such as bone marrow hematopoietic cells, to distinguish poor retrieval from a genuinely low signal in the study section (HPA: High in hematopoietic cells; standard IHC practice).

Could fixation explain weak or uneven BCLAF1 staining?

The selected paraffin-section caption does not state its fixative, so target-specific fixation sensitivity for BCLAF1 is unknown (datasheet A04100-3). Record the fixative, fixation duration, and section processing for each sample before comparing staining across batches (standard IHC practice). If staining varies, compare similarly processed sections in the same run and check whether nuclear detail and other routine tissue features are preserved (standard IHC practice; HPA: ubiquitous nuclear expression). Do not attribute a weak result to a particular BCLAF1 modification or cellular location: the record lists multiple modified residues and nuclear localisation, but supplies no fixation-response experiment (UniProt Q9NYF8: modified residues and subcellular location).

Should BCLAF1 staining be nuclear, cytoplasmic, or punctate?

Expect predominantly nuclear staining in tissue sections, with the possibility of a speckled nuclear pattern where resolution permits (HPA: ubiquitous nuclear expression; HPA: enhanced nuclear-speckle localisation). Cytoplasmic localisation is also annotated, so assess a cytoplasmic signal against the nuclear pattern in the same cell population instead of rejecting it automatically (UniProt Q9NYF8: cytoplasm and nucleus). BCLAF1 has no annotated transmembrane segment, making an isolated crisp membrane outline a reason to inspect controls and detection background (UniProt Q9NYF8: topology; standard IHC practice). Compare intact cells away from section edges, and use a nuclear counterstain to establish whether chromogen lies within nuclei (standard IHC practice).

Can isoforms or epitope accessibility change the IHC pattern?

BCLAF1 has 4 annotated isoforms, but the supplied caption does not identify the antibody epitope or establish which isoforms it detects (UniProt Q9NYF8: isoforms; datasheet A04100-3). Check the antibody's epitope documentation before treating a staining difference as an isoform difference, and document any unknown coverage (standard IHC practice). The record also lists numerous modified residues, including phosphoserines at positions 102 and 104, without showing that they affect this antibody (UniProt Q9NYF8: modified residues). If a pattern changes across samples, first verify retrieval, section processing, and nuclear morphology under matched conditions; an isoform or modification explanation requires separate evidence (standard IHC practice).

How can I check BCLAF1 localisation by multiplex IF?

Use IF as a separate optimisation, because the selected antibody caption documents chromogenic staining of a paraffin section, not an IF result (datasheet A04100-3). Pair BCLAF1 with a validated marker for the cell type being examined; for example, a validated glial marker can help identify the glial cells reported as strongly stained in cerebral cortex (HPA: High in cerebral-cortex glial cells; standard IF practice). Choose fluorophores after checking tissue autofluorescence and channel bleed-through, and include single-stain controls for the multiplex panel (standard IF practice). Optimise permeabilisation for access to the intracellular nuclear epitope, then assess whether signal overlaps nuclei and resolves into speckles where imaging permits (UniProt Q9NYF8: nuclear localisation and no transmembrane segment; HPA: nuclear speckles; standard IF practice).

What should I check when DAB appears outside BCLAF1-positive nuclei?

Check a no-primary control alongside the test section to identify chromogen deposition from the detection workflow (standard IHC practice). The selected caption uses a peroxidase-conjugated secondary antibody and DAB; a peroxidase block is a general chromogenic IHC step, not evidence of BCLAF1-specific behaviour (datasheet A04100-3; standard IHC practice). If diffuse staining persists, assess blocking, washes, primary-antibody concentration, and whether tissue edges collect excess reagent; the documented primary concentration is 2 μg/ml (datasheet A04100-3; standard IHC practice). Compare the background with intact nuclear signal in a control tissue before assigning faint cytoplasmic colour to BCLAF1, which has both nuclear and cytoplasmic annotations (UniProt Q9NYF8: subcellular location; standard IHC practice).

How should I quantify BCLAF1 across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and scoring region before measuring, then score nuclear staining using the percentage of positive cells and, when intensity is reproducible, an H-score (standard IHC practice; HPA: ubiquitous nuclear expression). Report the nuclear positivity threshold, intensity categories, and number of evaluable cells or tissue area for each section (standard IHC practice). If cell abundance varies, normalise positive-cell counts to the number of cells in that population, or report positive-cell density per mm² of viable tissue (standard IHC practice). Keep retrieval, imaging or scoring thresholds, and chromogen development comparable across batches, and analyse cytoplasmic staining separately from the expected nuclear pattern (datasheet A04100-3: EDTA pH 8.0 retrieval; HPA: ubiquitous nuclear expression; standard IHC practice).

How do I distinguish genuine BCLAF1 signal from staining artefacts?

Give greatest weight to staining within intact nuclei in relevant cells, because tissue IHC reports ubiquitous nuclear expression and enhanced consistency with RNA expression (HPA: tissue profile and reliability). High staining in bone marrow hematopoietic cells or cerebral-cortex glial cells can support a run-level control, although neither establishes specificity by itself (HPA: High in those cell populations; standard IHC practice). Treat isolated membrane outlines cautiously because BCLAF1 lacks a transmembrane segment, and review strong edge staining or colour over necrotic areas against adjacent intact tissue (UniProt Q9NYF8: topology; standard IHC practice). If staining also appears in the no-primary control, investigate endogenous peroxidase or detection background before interpreting differences between samples (standard IHC practice).

Boster reagents

Best BCLAF1 / Bcl-2-associated transcription factor 1 IHC Antibodies

The catalog includes anti-BCLAF1 antibodies for tissue IHC and cell IF/ICC (catalog: applications), with mouse and rat brain IHC images and a HeLa cell IF image (image captions).

Real IHC data

Anti-BCLAF1 Antibody ®

Cat # A04100-3

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Real IHC data Immunohistochemistry of BCLAF1 in mouse brain tissue with BCLAF1 antibody at 5 μg/mL.

Anti-BCLAF1 Antibody

Cat # A04100-1

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A04100-3 will render with its mouse brain paraffin-section IHC image; its other captions show rat brain paraffin sections and HeLa cell IF (A04100-3 image captions). A04100-1 will render with its mouse brain IHC image, and its application list includes IHC-P (A04100-1 image caption; catalog: applications).

Which to pick: Choose A04100-3 for paraffin-section tissue IHC because its own caption documents the retrieval, blocking, antibody incubation and chromogenic detection conditions; the fixative is unreported (A04100-3 IHC caption). For IF/ICC, choose A04100-3 because its own caption documents HeLa cell IF; A04100-1 has no listed IF application (A04100-3 IF caption; catalog: applications). For cross-species planning, A04100-3 has mouse and rat brain IHC images and listed human, mouse and rat reactivity; M04100 is a rabbit monoclonal with the same listed species and IHC/IF applications, but no supplied images (A04100-3 IHC captions; catalog: reactivity, clone, applications and image alts).

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

References

UniProt Consortium. UniProt entry Q9NYF8 (BCLF1_HUMAN, Bcl-2-associated transcription factor 1).
Human Protein Atlas. BCLAF1 tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. BCLAF1 subcellular location (ICC-IF): Localized to the nuclear speckles..
Human Protein Atlas. BCLAF1 antibody validation summary (3 antibodies).
BCLAF1 Regulates Osteoarthritic Cartilage Degradation Through Interaction with LAMTOR2. International journal of biological sciences 2025 — PMC11844276.
Sorbaria sorbifolia flavonoid derivative induces mitochondrial apoptosis in human hepatoma cells through Bclaf1. Frontiers in pharmacology 2024 — PMC11496133.
BCLAF1-induced HIF-1α accumulation under normoxia enhances PD-L1 treatment resistances via BCLAF1-CUL3 complex. Cancer immunology, immunotherapy : CII 2023 — PMC10700218.
Role of BCLAF-1 in PD-L1 stabilization in response to ionizing irradiation. Cancer science 2021 — PMC8486183.
PubMed PMID:10330179 — UniProt-cited evidence.
PubMed PMID:8724849 — UniProt-cited evidence.
PubMed PMID:14574404 — UniProt-cited evidence.