CHAF1B / Chromatin assembly factor 1 subunit B · IHC design guide

Design Immunohistochemistry for CHAF1B

Plan CHAF1B paraffin-section IHC around nuclear staining in dividing cells (HPA tissue IHC). Bone marrow hematopoietic cells and tonsil germinal center cells show high staining (HPA tissue IHC); the catalog antibody’s IHC dilution is 1:100–1:300 (datasheet A30479).

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

Immunohistochemistry protocol sheet for CHAF1B (IHC for CHAF1B): expected localisation Nuclear staining in dividing cells (HPA tissue IHC); cytoplasmic in M phase (UniProt), antibody A30479, validated IHC image, and IHC protocol steps — Printable **CHAF1B** IHC protocol sheet — expected localisation Nuclear staining in dividing cells (HPA tissue IHC); cytoplasmic in M phase (UniProt), antibody A30479, controls and protocol steps. Open the full CHAF1B IHC guide →

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CHAF1B 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 in dividing cells (HPA tissue IHC); cytoplasmic in M phase (UniProt)
	Staining pattern	Mainly nuclear staining in dividing cells (HPA tissue IHC)
	Antigen retrieval	Tris-EDTA pH 8.0 HIER, heat-mediated (datasheet A30479)
	Positive control ⓘ	Bone marrow+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 paraffin-section fixation consistent. (standard IHC practice; not target-specific)
	Caveat	The proportion of dividing cells can change the stained fraction (HPA tissue IHC)
	Regulation	Staining tracks dividing cells (HPA tissue IHC)
	Isoform / epitope	No isoforms or processing are annotated (UniProt)

Section 1

Recommended CHAF1B IHC & IF Protocols

The catalog antibody’s IHC-P protocol (datasheet A30479) is paired with published CHAF1B IHC methods from 2 studies (PMC7249347; PMC12492380).

Recommended immunohistochemistry (IHC-P) protocol parameters
Sample	Paraffin-embedded Human brain tissue; fixative not specified (datasheet A30479)
Fixation	Image fixative and duration unreported (datasheet A30479); verify before use.
Sectioning	4–5 µm sections on charged slides (standard)
Deparaffinisation	Xylene, graded ethanol series to water (standard)
Antigen retrieval	Heat retrieval: Tris-EDTA pH 8.0 (datasheet A30479); 20 min, 95–100 °C (standard)
Peroxidase block	3% H2O2, 10 min, room temperature (standard)
Blocking	10% normal serum of the secondary host, 30 min, room temperature (standard)
Primary antibody	Rabbit anti-CHAF1B, 1:100 - 1:300 (datasheet A30479)
Primary incubation	Overnight at 4 °C (standard)
Detection	HRP-polymer secondary, DAB chromogen 5–10 min (standard)
Counterstain	Hematoxylin, blue, dehydrate and mount (standard)
Expected result	CHAF1B-positive staining in hematopoietic cells of bone marrow (HPA tissue IHC: High). HPA tissue profile: Nuclear expression mainly in dividing cells. No signal in the no-primary control.

💡Decision noteStart with heat-mediated Tris-EDTA retrieval at pH 8.0 (datasheet A30479); sodium citrate microwave retrieval is a published comparison condition (PMC12492380).

Section 2

What Is the Expected CHAF1B Staining Pattern?

CHAF1B is primarily nuclear, with cytoplasmic localization reported in M phase (UniProt Q13112: subcellular location). In paraffin sections, expect nuclear staining mainly in dividing cells (HPA: tissue IHC profile). HPA rates the tissue pattern Enhanced but reports medium consistency with RNA expression (HPA: tissue IHC reliability). CHAF1B has no transmembrane segment (UniProt Q13112: topology).

What am I looking at on my slide?
Distinct nuclear staining in bone marrow hematopoietic cells or tonsil germinal center cells.	This fits the reported high staining in those cell groups and the predominantly nuclear pattern (HPA: tissue IHC). Judge the stained cells and compartment together; a dark area alone does not establish the expected pattern (general IHC practice).
Predominantly cytoplasmic staining across a section, with little or no nuclear signal.	Recheck specificity and detection before calling it CHAF1B: HPA describes mainly nuclear staining (HPA: tissue IHC). UniProt reports cytoplasmic localization in M phase, so a cytoplasmic cell alone is not conclusive evidence of artefact (UniProt Q13112: subcellular location).
Strong staining in adipocytes or another cell group reported as not detected.	HPA reports adipocytes in adipose tissue as not detected (HPA: tissue IHC). Unexpected staining warrants comparison with a no-primary control and an expected positive section; cross-reactivity or endogenous detection activity are possible causes (general IHC practice).
Diffuse color over cells and surrounding tissue, without clear nuclear boundaries.	The pattern is difficult to score as CHAF1B because the expected tissue signal is mainly nuclear (HPA: tissue IHC). Check background with a no-primary control, then review blocking, washes and detection development (general IHC practice).
No signal in bone marrow hematopoietic cells or tonsil germinal center cells.	Both are reported high-staining groups (HPA: tissue IHC), making them useful positive comparators. A negative result may reflect the section, staining workflow or antibody performance; first confirm tissue identity and control staining (general IHC practice).

💡Expected CHAF1B appearanceCall a result consistent with CHAF1B when distinct nuclear staining occurs mainly in dividing cells, especially the high-staining groups in bone marrow or tonsil (HPA: tissue IHC); widespread staining without a nuclear pattern calls for control review (general IHC practice).

How each factor affects the staining
Cell type and tissue	High staining is reported in bone marrow hematopoietic and tonsil germinal center cells; several other groups are medium or low (HPA: tissue IHC). Compare like cell types when assessing intensity (general IHC practice).
Cell-cycle context	HPA describes nuclear expression mainly in dividing cells (HPA: tissue IHC); UniProt notes DNA replication foci and cytoplasmic localization in M phase (UniProt Q13112: subcellular location). A single cell need not represent the whole section.
Antibody validation	HPA lists HPA021679 as IHC Approved and CAB033604 as IHC Enhanced (HPA: antibody validation). The tissue profile is rated Enhanced with medium staining-to-RNA consistency (HPA: tissue IHC reliability); interpret unexpected patterns with controls.
Protein features	CHAF1B has no transmembrane segment, signal peptide, propeptide or annotated isoform in the supplied record (UniProt Q13112: topology and processing). These features do not identify an antibody epitope or establish fixation sensitivity.
IF/ICC Q: Where should fluorescence appear?	A: HPA supports nucleoplasmic localization in ICC-IF (HPA: subcellular). Use this as a localization cross-check; the fluorescence result does not establish an IHC retrieval condition (general IHC practice).

Why is my staining missing, weak or wrong?
Situation	Likely cause	Next action
Expected positive tissue is blank.	Section identity, antibody exposure or detection may have failed (general IHC practice).	Confirm bone marrow hematopoietic or tonsil germinal center cells are present (HPA: High); check reagent sequence, retrieval against the antibody's IHC instructions, and detection controls (general IHC practice).
Nuclear signal is faint in a reported high-staining group.	Weak assay signal or few stained cells may make assessment uncertain (general IHC practice).	Inspect the expected cell group at higher magnification, compare a positive section, and adjust the validated staining workflow within its instructions (general IHC practice; HPA: High in bone marrow and tonsil groups).
Staining is mostly cytoplasmic.	Diffuse artefact is possible, although UniProt reports cytoplasmic CHAF1B in M phase (UniProt Q13112: subcellular location).	Assess whether nuclear staining is also present; compare positive and no-primary controls before assigning the cytoplasmic signal to CHAF1B (HPA: mainly nuclear; general IHC practice).
Reported negative adipocytes stain strongly.	Cross-reactivity or endogenous detection activity is possible (general IHC practice; HPA: adipocytes in adipose tissue not detected).	Check a no-primary control and the positive tissue, then review blocking and the detection system (general IHC practice).
Color covers the section without cell-level definition.	Background from detection development, insufficient washes or nonspecific binding is possible (general IHC practice).	Compare a no-primary control; review blocking, washes and development, then score only resolved cell-associated staining (general IHC practice).
A medium- or low-staining cell group looks negative.	The reported signal may be harder to distinguish from background than in a high-staining comparator (HPA: tissue IHC levels; general IHC practice).	Check a high-staining bone marrow or tonsil group in the same run before concluding the assay failed; record the examined cell type (HPA: High; general IHC practice).

Source: UniProt Q13112 Human Protein Atlas tissue Human Protein Atlas subcellular

Experiment evidence

IHC & IF images showing CHAF1B staining

Immunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tris-EDTA,pH8.0 was used for antigen retrieval. Negetive contrl (right) obtaned from antibody was pre-absorbed by immunogen peptide.

A30479 · IHCImmunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tr

HPA · Cerebral cortexHuman Protein Atlas IHC, Cerebral cortex. Source: https://www.proteinatlas.org/ENSG00000159259-CHAF1B/tissue/cerebral+cortex#imid_13260895

HPA · ColonHuman Protein Atlas IHC, Colon. Source: https://www.proteinatlas.org/ENSG00000159259-CHAF1B/tissue/colon#imid_13261685

HPA · LiverHuman Protein Atlas IHC, Liver. Source: https://www.proteinatlas.org/ENSG00000159259-CHAF1B/tissue/liver#imid_13261686

HPA · KidneyHuman Protein Atlas IHC, Kidney. Source: https://www.proteinatlas.org/ENSG00000159259-CHAF1B/tissue/kidney#imid_13261687

HPA · ICC-IF A-431Human Protein Atlas immunofluorescence, A-431, antibody HPA021679; green = CHAF1B, blue = nucleus, red = microtubules.

← swipe / scroll for more images →

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

Sample controls for CHAF1B IHC & IF

🧪Run tonsil first and score germinal center cells for nuclear staining (HPA: High in tonsil germinal center cells; HPA subcellular: nucleoplasm supported). Use adipose tissue as the negative, focusing on adipocytes (HPA: Not detected in adipocytes); on the tonsil slide, record cells without nuclear staining as internal negatives without assuming every cell outside the germinal center is negative.

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

Negative control tissue: Adipose tissue (HPA Not detected)

ICC-IF cell lines (HPA subcellular resource): HPA ICC-IF images show CHAF1B in A-431, U-251MG, U2OS, U2OS, siRNA 1 (10x), U2OS, siRNA 1 (40x), U2OS, scrambled (10x), U2OS, scrambled (40x), with annotated localisation: Nucleoplasm (supported) (HPA subcellular).

Technical controls: Include a no-primary (secondary-only) control and nonspecific IgG matched to the primary antibody’s host species, with subclass matching if it is monoclonal (standard IHC practice). Check specificity with CHAF1B knockout material or immunogen-peptide preabsorption (A30479 tissue-IHC caption: peptide-absorbed control), and quench endogenous peroxidase in the tonsil section before chromogenic detection (standard IHC practice).

⚠️Feasibility: A CHAF1B-specific fixation window or fixation effect is unreported, and the selected A30479 paraffin-section caption does not state a fixative (A30479 tissue-IHC caption). Retrieval dependence is unestablished; the caption reports high-pressure, high-temperature Tris-EDTA retrieval at pH 8.0 for a brain section, which is a condition to assess on tonsil (A30479 tissue-IHC caption). Whether frozen sections are easier is unreported; IF/ICC images support nucleoplasmic localization (HPA subcellular: nucleoplasm supported), while endogenous peroxidase in inflammatory cells can complicate chromogenic scoring in tonsil (standard IHC practice).

HPA tissue IHC evidence for CHAF1B

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
Bone marrow	Hematopoietic cells	High	Protein (IHC)	HPA →
Tonsil	Germinal center cells	High	Protein (IHC)	HPA →
Appendix	Lymphoid tissue	Medium	Protein (IHC)	HPA →
Duodenum	Glandular cells	Medium	Protein (IHC)	HPA →
Esophagus	Squamous epithelial cells	Medium	Protein (IHC)	HPA →

Undetected expression · recommended negative controls

Tissue	Cell type	Level	Evidence	Source
Adipose tissue	Adipocytes	Not detected	Protein (IHC)	HPA →
Adrenal gland	Glandular cells	Not detected	Protein (IHC)	HPA →
Breast	Adipocytes	Not detected	Protein (IHC)	HPA →
Caudate	Glial cells	Not detected	Protein (IHC)	HPA →
Cerebellum	Cells in granular layer	Not detected	Protein (IHC)	HPA →

Section 3

Advanced CHAF1B IHC Tips

Troubleshoot CHAF1B chromogenic IHC by checking nuclear staining, section processing, controls, and scoring against the reported tissue pattern.

What retrieval conditions should I try first when CHAF1B nuclear staining is weak?

Use heat-mediated Tris-EDTA at pH 8.0 for antigen retrieval in this paraffin-section assay (datasheet A30479). The selected tissue image used high pressure and temperature for retrieval, followed by antibody at 1:100 overnight at 4°C (caption A30479). If nuclear signal remains weak, compare shorter and longer heating on adjacent sections while holding dilution and detection constant; assess tissue morphology after each condition (standard IHC practice). Evaluate signal in dividing-cell regions, where nuclear expression is expected (HPA: nuclear expression mainly in dividing cells), and include the peptide-preabsorbed control used for the selected image (caption A30479).

How should I troubleshoot fixation-related loss of CHAF1B staining?

The selected image identifies paraffin-embedded human brain but does not report its fixative, so CHAF1B-specific fixation sensitivity is unknown (caption A30479). Record the fixative, fixation duration, and processing history for each section before comparing staining results (standard IHC practice). If similarly processed sections stain differently, run them together with the same pH 8.0 Tris-EDTA retrieval and detection conditions to isolate processing as a possible cause (datasheet A30479; standard IHC practice). Judge whether nuclear detail and overall morphology remain intact, and use the same known staining control across runs before attributing weak signal to fixation (standard IHC practice).

Should CHAF1B staining be nuclear or cytoplasmic in paraffin sections?

Expect predominantly nuclear staining, especially in dividing cells, when reading chromogenic tissue sections (HPA: nuclear expression mainly in dividing cells). A nucleoplasmic pattern is supported by cell imaging, while UniProt also reports DNA replication foci and cytoplasmic localization during M phase (HPA: nucleoplasm supported; UniProt Q13112: subcellular location). Examine whether a cytoplasmic signal is confined to morphologically mitotic cells before treating it as plausible CHAF1B staining (UniProt Q13112: cytoplasmic in M phase; standard IHC practice). Diffuse cytoplasmic color across many quiescent cells should prompt review of blocking, antibody concentration, and the negative control rather than a change in the expected tissue pattern (HPA: nuclear expression mainly in dividing cells; standard IHC practice).

Could isoforms or epitope masking explain inconsistent CHAF1B staining?

The supplied record lists 0 annotated isoforms and one chain spanning residues 1–559, so an isoform-specific explanation is unsupported here (UniProt Q13112: isoforms and processing). It does not map this antibody’s epitope, preventing a claim that retrieval exposes a particular region (record: epitope not supplied). CHAF1B has no transmembrane segment and has annotated phosphorylation sites, including residues 394 and 409; an overlapping epitope could, in principle, respond to modification, but overlap is unknown (UniProt Q13112: topology and modified residues). Compare retrieval and dilution on matched sections, then assess nuclear signal with the peptide-preabsorbed control before proposing an epitope mechanism (datasheet A30479; caption A30479; standard IHC practice).

How can I check CHAF1B localization in a parallel IF experiment?

Use IF as a separate localization check: cell imaging supports nucleoplasmic CHAF1B and includes U2OS scrambled and siRNA image sets, without supplying fixation or antibody performance details for this assay (HPA: subcellular). Multiplex with a marker identifying the expected dividing-cell population, and choose spectrally separated fluorophores, favoring a far-red CHAF1B channel if the specimen has shorter-wavelength autofluorescence (HPA: nuclear expression mainly in dividing cells; standard IF practice). Use controlled permeabilization to give antibody access to the nuclear epitope; CHAF1B has no transmembrane segment, and the antibody’s epitope is unmapped (UniProt Q13112: topology; record: epitope not supplied). Check single-color and secondary-only controls before comparing IF localization with chromogenic nuclear staining (standard IF practice; HPA: nuclear expression mainly in dividing cells).

What should I check when CHAF1B IHC shows widespread brown background?

First compare nuclear color with the peptide-preabsorbed negative control shown for the selected antibody image (caption A30479). Review peroxidase blocking, secondary-reagent controls, wash stringency, and DAB development time; these are general chromogenic IHC controls rather than CHAF1B-specific findings (standard IHC practice). If background rises with the reported 1:100 overnight incubation at 4°C, titrate the antibody while keeping retrieval at Tris-EDTA pH 8.0 and development conditions fixed (caption A30479; datasheet A30479; standard IHC practice). Give greatest weight to discrete nuclear staining in appropriate cells, since tissue profiling describes nuclear expression mainly in dividing cells (HPA: tissue profile).

How should I quantify CHAF1B staining across paraffin sections? ⚠ ANSWER MARKED FOR VERIFICATION

Define the cell population and tissue region before scoring, because CHAF1B nuclear expression is reported mainly in dividing cells (HPA: tissue profile). For chromogenic sections, record the percentage of positive nuclei and an H-score using intensity grades 0–3, yielding a possible 0–300 range (standard IHC practice). When tissue composition varies, also report positive nuclei per mm² and normalize counts to all evaluable nuclei within the same annotated compartment (standard IHC practice). Keep retrieval, imaging, threshold, and exclusion rules consistent, and exclude folds, necrosis, and section edges from the denominator (datasheet A30479: retrieval; standard IHC practice).

How do I distinguish true CHAF1B staining from tissue artefact?

A credible positive pattern is predominantly nuclear in an appropriate proliferating population, consistent with the reported tissue profile and supported nucleoplasmic localization (HPA: tissue profile and subcellular). High staining in bone-marrow hematopoietic cells and tonsil germinal-center cells offers reference patterns, while the selected brain image includes a peptide-preabsorbed control (HPA: tissue IHC; caption A30479). Treat color confined to section edges, necrotic areas, or sites of residual endogenous peroxidase as suspect and compare it with negative controls (standard IHC practice). Widespread cytoplasmic staining in nonmitotic cells warrants review, although cytoplasmic CHAF1B can occur during M phase (UniProt Q13112: subcellular location; standard IHC practice).

Boster reagents

Best CHAF1B / Chromatin assembly factor 1 subunit B IHC Antibodies

A30479 has a human brain paraffin-section IHC image (A30479 IHC caption) and lists IF/ICC use in human, mouse and rat (catalog applications/reactivity).

Real IHC data Immunohistochemical analysis of paraffin-embedded Human brain. Antibody was diluted at 1:100(4° overnight). High-pressure and temperature Tris-EDTA,pH8.0 was used for antigen retrieval. Negetive contrl (right) obtaned from antibody was pre-absorbed by immunogen peptide.

Anti-CAF1B CHAF1B Antibody

Cat # A30479

View product →

A30479 is the sole SKU in the rendered cards (cards payload). Its own IHC caption documents paraffin-embedded human brain; the catalog also lists IHC, IF and ICC applications for human, mouse and rat (A30479 IHC caption; catalog applications/reactivity).

Which to pick: For tissue IHC, choose A30479: its image documents human brain paraffin sections at 1:100 with Tris-EDTA pH 8 retrieval; the fixative is unreported (A30479 IHC caption). For human IF/ICC, M03959-1 offers rabbit monoclonal clone 20C00 with both applications listed, although the payload supplies no IF image (catalog clone/applications/IF images). For cross-species IHC or IF/ICC, A30479 is the rabbit polyclonal option listed for human, mouse and rat; its IF/ICC dilution range is 1:200–1:1000 (catalog dilution_raw/reactivity/applications).

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

References

UniProt Consortium. UniProt entry Q13112 (CAF1B_HUMAN, Chromatin assembly factor 1 subunit B).
Human Protein Atlas. CHAF1B tissue IHC expression (reliability: Enhanced).
Human Protein Atlas. CHAF1B subcellular location (ICC-IF): Localized to the nucleoplasm..
Human Protein Atlas. CHAF1B antibody validation summary (2 antibodies).
USP4-Dependent CHAF1B Stabilization Regulates Distinct SETDB1 Ubiquitin States Linked to AKT T308 Signaling and Lipogenic Remodeling in HCC. Advanced science (Weinheim, Baden-Wurttemberg, Germany) 2026 — PMC13624420.
Ubiquitin ligase CHAF1B induces cisplatin resistance in lung adenocarcinoma by promoting NCOR2 degradation. Cancer cell international 2020 — PMC7249347.
CHAF1B: the hidden culprit behind sorafenib resistance in hepatocellular carcinoma. International journal of medical sciences 2025 — PMC12492380.
The histone chaperone CAF-1 safeguards somatic cell identity. Nature 2015 — PMC4866648.
PubMed PMID:7600578 — UniProt-cited evidence.
PubMed PMID:10830953 — UniProt-cited evidence.
PubMed PMID:15489334 — UniProt-cited evidence.