Journal: Investigative Ophthalmology & Visual Science

Article Title: Bipolar Cell Type-Specific Expression and Conductance of Alpha-7 Nicotinic Acetylcholine Receptors in the Mouse Retina

doi: 10.1167/iovs.18-25753

Figure Lengend Snippet: Immunohistochemistry Antibodies and Toxins

Article Snippet: HCN4 clone N114/10 , Fusion protein amino acids 1019-1108 at C-terminus of rat HCN4 , NeuroMab (Davis, CA, USA), 75-150, Mouse monoclonal , AB_2248534 , 1:200.

Techniques: Immunohistochemistry

Journal: Investigative Ophthalmology & Visual Science

Article Title: Bipolar Cell Type-Specific Expression and Conductance of Alpha-7 Nicotinic Acetylcholine Receptors in the Mouse Retina

doi: 10.1167/iovs.18-25753

Figure Lengend Snippet: Type 2, but not type 3, OFF bipolar cells possessed α7-nAChRs. (A) Type 2 bipolar cells were identified with Syt2 (green), which showed αBgTx fluorescence (magenta, 32/39 cells). (B) Type 2 bipolar cells depolarized in response to PNU282987 puff (left). Individual traces shown in gray, and average trace displayed in black. A NB-filled cell depolarized in response to PNU, which was confirmed by Syt2 staining (right). (C) HCN4-labeled type 3a (green), which were partially visualized with αBgTx fluorescence (magenta) (20/89 cells). (D) PKAIIβ-labeled type 3b bipolar cells (green), which were not labeled with αBgTx fluorescence (magenta) (0/15 cells) PKAIIβ-labeled various amacrine cells that localize along the outer border of the INL. Scale bar: (A–C) 10 μm, (D) 20 μm. Images are maximum intensity projections of multiple slice sections.

Article Snippet: HCN4 clone N114/10 , Fusion protein amino acids 1019-1108 at C-terminus of rat HCN4 , NeuroMab (Davis, CA, USA), 75-150, Mouse monoclonal , AB_2248534 , 1:200.

Techniques: Fluorescence, Staining, Labeling

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Colocalization of Thy1 and HCN4 in ganglion cell layer somata. (A–C) Single optical section through the ganglion cell layer of a retina incubated in polyclonal anti-HCN4 primary and DyLight 488-conjugated secondary, anti-Thy1 primary, and DyLight 549-conjugated secondary, and Nissl stain. Fluorescence from DyLight 488 (A), DyLight 549 (B), and Nissl (C) assigned to the green, red, and blue color channels, respectively, and merged in (C). (D–F) Single optical section through the ganglion cell layer of a different portion of the same retina, processed as in (A–C) except that the anti-HCN4 primary was preincubated with immunogen. The scale bar in (C) (20 μm) applies to (C) alone; the scale bar in (F) (20 μm) applies to (A, B, D–F).

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, Staining, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Colocalization of Thy1 and HCN4 in ganglion cell somata. (A–C) A single optical section through the ganglion cell layer of a retina incubated in monoclonal anti-HCN4 primary and DyLight 488-conjugated secondary (A, C), anti-Thy1 primary and DyLight 549-conjugated secondary (B, C), and anti-Brn3a primary and DyLight 649-conjugated secondary (C). Fluorescence from DyLight 488, 549, and 649 were assigned to the green, red, and blue color channels, respectively, and are merged in (C). (D–F) Single optical section through the ganglion cell layer of a different portion of the same retina, processed as in (A–C) except that the anti-Thy1 primary was not applied. The scale bar in (C) (20 μm) applies to (C) alone; the scale bar in (F) (20 μm) applies to (A, B, D–F).

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, Fluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Coimmunoprecipitation of Thy1 and HCN4 by beads coated with anti-Thy1 antibody. Protein G-coated beads (Dynabeads, 100.03D; Invitrogen) were BS3-cross-linked to Thy-1 antibody and then incubated with lysate. Proteins were eluted from these beads, analyzed with Western blot, and sequentially probed for Thy1 (lanes a–d), HCN4 (lanes e–h) using polyclonal antibody, and ABCR (lanes i–l). Before the second and third probes, the previously applied primary antibodies were stripped with glycine. The material aliquoted into each SDS-PAGE gel lane is specified underneath by the combination of + and −. Lanes a, e, and i are material eluted from raw beads (without antibody coating and without lysate). Lanes b, f, and j are material eluted from antibody-coated beads without lysate. Lanes c, g, and k are proteins eluted from Thy1 antibody-coated beads incubated with lysate. Lanes d, h, and l are lysate alone. The migration distances of the molecular weight standards along the left apply to the lanes probed by Thy1 antibody. Those along the right apply to the lanes probed by HCN4 and ABCR antibodies.

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, Western Blot, SDS Page, Migration, Molecular Weight

Journal: Investigative Ophthalmology & Visual Science

Article Title: Thy1 Associates with the Cation Channel Subunit HCN4 in Adult Rat Retina

doi: 10.1167/iovs.11-9307

Figure Lengend Snippet: Coimmunoprecipitation of Thy1 and HCN4 by incubation of beads, coated with monoclonal anti-HCN4 antibody, with lysates, formatted as in Figure 4. The proteins eluted from these beads were separated by SDS-PAGE, with samples of the lysate and molecular weight standards in adjacent lanes. The gels were analyzed with Western blot and the blots were separated into three segments by slicing at the level of the 185 kD standard and at approximately 50 kD. The upper, middle, and lower segments were probed with antibodies against ABCR, HCN4 (polyclonal), and Thy1, respectively. The ABCR and Thy1 antibodies were visualized with Fc fragment-specific, DyLight 649-conjugated, goat anti-mouse IgG. The HCN4 antibody was visualized with detection reagent (Clean-Blot IP; Thermo Scientific).

Article Snippet: The other anti-HCN4 antibody was clone N114/10 (73-150; UC Davis/NIH NeuroMab Facility, Davis, CA), a monoclonal mouse IgG1 raised against a fusion protein of GST and amino acids 1019 to 1108 of rat HCN4.

Techniques: Incubation, SDS Page, Molecular Weight, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: Age-dependent down-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 4 causes deterioration of canine sinoatrial node function.

doi: 10.1093/abbs/gmx026

Figure Lengend Snippet: Figure 5. The mRNA expression levels of ADRB1, CACNA1C, HCN4, and KCNE2 in the SAN of dogs HCN4 mRNA was significantly decreased in the aged group compared with those in the young and adult groups (n = 4 per group). *P < 0.05 adult group vs. aged group ; #P < 0.05 young group vs. aged group.

Article Snippet: The membranes were incubated with the primary antibodies, anti-HCN4 antibody (1:500; Boster Biotechnology, Wuhan, China), and antiGAPDH antibody (1:500; Boster Biotechnology), overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:1000, Zhong Shan Jin Qiao Biotechnology, Beijing, China) secondary antibody for 1 h at 37°C.

Techniques: Expressing

Journal: Acta biochimica et biophysica Sinica

Article Title: Age-dependent down-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 4 causes deterioration of canine sinoatrial node function.

doi: 10.1093/abbs/gmx026

Figure Lengend Snippet: Figure 6. HCN4 expression in cardiac pacemaker cells within SAN detected by IF staining Immunofluorescent analysis of HCN4 (red) protein expression in fro- zen sections of young (A–C), adult (D–F), and aged dogs (G–I). The expression of HCN4 protein within the SAN declines with aging.

Article Snippet: The membranes were incubated with the primary antibodies, anti-HCN4 antibody (1:500; Boster Biotechnology, Wuhan, China), and antiGAPDH antibody (1:500; Boster Biotechnology), overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:1000, Zhong Shan Jin Qiao Biotechnology, Beijing, China) secondary antibody for 1 h at 37°C.

Techniques: Expressing, Staining

Journal: Acta biochimica et biophysica Sinica

Article Title: Age-dependent down-regulation of hyperpolarization-activated cyclic nucleotide-gated channel 4 causes deterioration of canine sinoatrial node function.

doi: 10.1093/abbs/gmx026

Figure Lengend Snippet: Figure 7. HCN4 protein expression in the SAN of dogs detected by western blot analysis (A) HCN4 protein expression decreased gradually from the young to the aged group. (B) Comparison of the mean OD value indicates that the expression of HCN4 declined with increasing age. n = 4 per group. *P < 0.05 young group vs. adult group; #P < 0.05 adult group vs. aged group.

Article Snippet: The membranes were incubated with the primary antibodies, anti-HCN4 antibody (1:500; Boster Biotechnology, Wuhan, China), and antiGAPDH antibody (1:500; Boster Biotechnology), overnight at 4°C, followed by incubation with horseradish peroxidase-conjugated anti-rabbit or anti-goat IgG (1:1000, Zhong Shan Jin Qiao Biotechnology, Beijing, China) secondary antibody for 1 h at 37°C.

Techniques: Expressing, Western Blot, Comparison