Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: H1fx is broadly and strongly expressed in E6.0–7.0 embryos. (A–E) The sagittal sections of E6.0 (TS8) embryos at the advanced egg cylinder stage were stained with anti-H1fx (A), H1f0 (B), E-cadherin (C), Ki-67 (D) and DAPI (E). The epiblast (eb) and extraembryonic ectoderm (ex) were surrounded by visceral endoderm (ven). (F–O) The sagittal sections of E7.0 (TS 10) embryos at the late streak, early allantoic bud stage, were stained with anti-H1fx (F and K), H1f0 (G and L), E-cadherin (H and M), Ki-67 (I and N), and DAPI (J and O). The lower panels (K–O) are high-magnification images of the upper ones (F–J). Yellow arrows represent trophoblasts. White arrows and white arrowheads indicate the node and allantoic bud, respectively. Scale bars: A to J, 50 µm; K to O, 25 µm. Abbreviations: epc, ectoplacental cone; am, amnion; en, endoderm; me, mesoderm; ec, ectoderm.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: Expression of H1fx is further restricted at E14.5 (TS23). Sagittal sections of an E14.5 (TS23) embryo were stained with anti-Ki-67 (A, C, E, H, K, and M), H1fx (B, D, F, I, L, and N), tyrosine hydroxylase [TH; (G)], H1f0 (J), and TUJ1 (O). (A and B) The paramedian plane including the corpus striatum of the lateral ventricle (large white arrows), spinal cord (sc), heart (h), liver (liv), lung (lu), kidney (ki), and midgut (small white arrows). (C and D) Bottom of the third ventricle (3v) with posterior part of the pituitary (black arrowheads) and Rathke’s pouch (rp). (E–G) The thoracic part includes the cartilage-primordium (cp), lung (lu), and sympathetic ganglia (sg). (H–J) The pancreatic primordium includes the tubular exocrine tissues (ex) and endocrine tissues (enc). (K and L) The adrenal primordium (ad) contains the H1fx-positive cells (black arrows), kidney (ki) with early nephrons (white arrowheads), and liver (liv). (M–O) The cross sections of the midgut tube contain the endodermal epithelium (ep) surrounding the lumen, smooth muscle layer (sm), and enteric neural cells (black arrows). Scale bars: A and B, 100 µm; C to L, 100 µm. Abbreviation: ea, ear.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Expressing, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: H1fx is locally expressed in the P56 adult brain. Coronal sections of the P56 brain were stained with Ki-67 (A, F, K and, O), H1fx (B, G, L, and P), nestin (C, H, and Q), NeuN (D, I, M, and R), and H1f0 (E, J, and N). (A–E) Arrows represent the subventricular zone along with the lateral ventricles (lv). (F–J) High-magnification images of the boxed regions in A to E, respectively. The high-magnification image of the boxed region in H was indicated as the inset. Black arrowheads indicate the nestin-positive cells. (K–N) The cerebral cortex and the hippocampus. The inset panels in K to N are high-magnification images of the granular zone, including the subgranular zone (sgz). (O–R) Coronal sections of the cerebellum at P56. The inset panel in P represents an enlarged image of the granular layer (gra) including Purkinje cells (pc) and granular cells (white arrowheads). (S) H1fx mRNA expression was analyzed by quantitative real-time PCR (n=4, for samples 1–4, and n=3, for samples 5 and 6). (T) H1fx protein expression was analyzed by western blot. The triplicated samples for each condition were examined, and the representative result was shown. (U) The quantification of the western blot of H1fx. The signal intensities were quantified by Image Analyzer, and the H1fx values were normalized by histone H3 as an internal standard of nuclear protein. (V) The western blot results of H1f0 with the same samples as T. The samples examined in T to V are as follows: lane 1, E12.5 brain; lane 2, E15.5 brain; lane 3, P0 cerebrum; lane 4, P7 cerebrum; lane 5, P56 cerebrum; lane 6, P56 colon. *p<0.05, **p<0.01 (vs E12.5-br, two-tailed Student’s t-test). Scale bars: A to E, O to R, 500 µm; F to J, 50 µm; K to N, 200 µm. Abbreviation: cc, cerebral cortex; svz, subventricular zone.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Staining, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: H1fx is expressed at the neural tube, somites, mesenchymal cells, and endoderm during the somitogenesis stage. (A–E) Sagittal sections of E8.0 (TS 12) embryos. (F–M) Sagittal sections of E8.5 (TS 13) embryos. Sections were stained with anti-H1fx (A, F, and J), H1f0 (B, G, and K), Ki-67 (C, H, and L), E-cadherin (D), DAPI (E and M), and Sox2 (I). The inset panels in A and B show enlarged images of a portion of embryos. (F–I) The white and black arrowheads represent the neural tube and somites, respectively. Black arrows represent the area of septum transversum. (J–M) The caudal part includes somites (so) and the septum transversum (white arrows). Scale bars: A to E, 100 µm; F to I, 200 µm; J to M, 50 µm. Abbreviations: epc, ectoplacental cone; ecc, exocoelomic cavity; am, amnion; nu, neural tube; ys, yolk sac; h, heart rudiment.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: H1fx is strongly expressed in the brain and spinal cord at the early organogenesis stage. (A–D) The sagittal sections were stained with anti-H1fx (A), H1f0 (B), Ki-67 (C), and DAPI (D). Arrowheads represent the branchial arch. (E–H) The abdominal region containing the alimentary canal was stained with anti-H1fx (E) and anti-Sox10 (F), and their merged image is also shown (G). Yellow arrows indicate the mesenchymal tissues. (H) Enlargement of the boxed area is shown in panel G. White arrows represent the double-stained neural crest cells. Scale bars A to D, 500 µm; E to G, 200 µm; H, 50 µm. Abbreviations: br, brain; sc, spinal cord; h, heart; se, septum transversum; es, esophagus; lub, lung bud; gt, gut; ao, aorta.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: H1fx expression is localized at E12.5 (TS21) during the organogenesis stage. The transverse sections of E12.5 embryos were stained with anti–Ki-67 (A, D, G, J, M, and P), H1fx (B, E, H, K, N, and Q), tyrosine hydroxylase (TH; I, O, and R), H1f0 (L), control rabbit IgG (C), and control mouse IgG (F): (A–C) head, containing the fourth ventricle (4v) and lateral ventricle of the forebrain; (D–F) face, containing the spinal cord (sc), eye (black arrowheads), inner ear (ea), and facial mesenchymal tissue (fm); (G–I) thorax, containing the esophagus (es), trachea (tr), heart (h), pre-cartilage condensation (pcc), dorsal root ganglia (drg), and sympathetic ganglia (black arrows); (J–L) abdominal, containing the lung (lu), liver (liv), gut (white arrows), pcc, and stomach (st); (M–R) caudal region, containing the liver, stomach, gut, sympathetic ganglia (black arrows) pcc, and abdominal aorta (asterisks); and (P-R) high-magnification images of the boxed areas in panels M, N, and O, respectively. H1fx was observed at the mesonephric tubules (white arrowheads), sympathetic ganglia (black arrows), and adrenal primordium (ad). The peripheral zone of the gonad (gn) was positive for H1fx. (S) The positions of sections in the embryo from the head to caudal region are schematically indicated by I to V. Scale bars: A to O, 500 µm; P to R, 50 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Expressing, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Temporally and Spatially Regulated Expression of the Linker Histone H1fx During Mouse Development

doi: 10.1369/0022155417723914

Figure Lengend Snippet: Expression of H1fx and H1f0 in the other adult tissues. Sections from P56 mice are stained with H1fx (A, D, and G), H1f0 (B, E, and J), and TUJ1 (H). (A–C) The pancreas is composed of the endocrine part, the islets of Langerhans (ln), and the exocrine tissues (exc). (D–F) Adrenal gland consisting of the cortex (ctx) and the medulla (me). (G–J) Cross section of the small intestine, consisting of the mucosa (mu), submucosa (smu), and muscularis. White arrows represent the H1fx- and TUJ1-positive cells. (I) Merged image of H1fx (G), TUJ1 (H), and DAPI. Scale bars: A to J, 50 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-H1X (anti-H1fx) polyclonal antibody (1:1500; Abcam, Cambridge, UK), mouse anti–Ki-67 monoclonal antibody (1:80; BD Biosciences, San Jose, CA), rabbit anti–E-cadherin polyclonal antibody (1:200; Cell Signaling Technology, Danvers, MA), mouse anti-Sox2 monoclonal antibody (1:600; Merck Millipore, Darmstadt, Germany), mouse anti-nestin monoclonal antibody (1:500; eBioscience, San Diego, CA), mouse anti-Sox10 monoclonal antibody (1:200; eBioscience), mouse anti-tyrosine hydroxylase monoclonal antibody (1:800; Merck Millipore), mouse anti-neuronal Nuclei (NeuN) monoclonal antibody (1:600; Merck Millipore), mouse anti-tubulin β-3 (TUJ1) monoclonal antibody (1:2500; BioLegend, San Diego, CA), rabbit anti-histone H1.0 (anti-H1f0) polyclonal antibody (1:500; GeneTex, Irvine, CA), and rabbit anti-calretinin (CR) polyclonal antibody (1:2500; Swant, Switzerland).

Techniques: Expressing, Staining