Journal: Neural Regeneration Research

Article Title: Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

doi: 10.4103/1673-5374.353852

Figure Lengend Snippet: Residual RGCs and axons had survived at 4 weeks after ONC. (A) The decrease in RGCs after ONC. Dual-color staining of the central, mid-peripheral, and peripheral retinas via an intravitreal injection of CTB (Alexa Fluor 488, green, labeling axons) and RBPMS (Alexa Fluro 647, gray, labeling RGCs) in the control and ONC groups. Scale bars: 50 µm. (B) The decrease in retrograde nerve fibers after ONC. Single channel of CTB staining from A. Scale bars: 50 µm. (C) The decrease in total nerve fibers after ONC. Immunostaining of the central, mid-peripheral, and peripheral retinas with NF-L staining (Alexa Fluro 488, gray, labeling nerve fibers). Scale bars: 50 µm. (D–F) Quantification of the numbers of RGCs ( n = 4 eyes per group), CTB fluorescence density (control: n = 4 eyes ONC: n = 3 eyes), and NF-L fluorescence density ( n = 3 eyes each group) in the control and ONC groups. (G) The decrease in axons after ONC. Axon immunostaining in the central and peripheral optic nerves via GAP-43 (Alexa Fluor 488, green, labeling axons) and DAPI (blue, labeling nucleus) in the control and ONC groups ( n = 3 per group); the central and peripheral regions are further magnified (gray) in the yellow boxes. Scale bars: 50 µm. (H) Quantification of the GAP-43 fluorescence density in the control and ONC groups ( n = 3 per group). Data from three independent experiments are presented as the mean ± SEM. * P < 0.05, ** P < 0.01, **** P < 0.0001 (two-way analysis of variance followed by Sidak’s multiple comparisons). CTB: Cholera toxin B subunit; DAPI: diamidino-phenyl-indole; GAP-43: growth-associated protein-43; NF-L: neurofilament-light chain; ONC: optic nerve crush; RBPMS: RNA binding protein with multiple splicing; RGCs: retinal ganglion cells.

Article Snippet: After three washes with PBS-0.1% Tween 20, goat anti-rabbit Alexa Fluor 488 (1:500, CST, Cat#4412S, RRID: AB_1904025) or goat anti-mouse Alexa Fluor 647 (1:500, CST, Cat#4410, RRID: AB_1904023) was applied.

Techniques: Staining, Injection, Labeling, Immunostaining, Fluorescence, RNA Binding Assay

Journal: Neural Regeneration Research

Article Title: Use of a tissue clearing technique combined with retrograde trans-synaptic viral tracing to evaluate changes in mouse retinorecipient brain regions following optic nerve crush

doi: 10.4103/1673-5374.353852

Figure Lengend Snippet: Retrograde labeling of RGCs in ONC eyes. (A, B) Comparison of different dose of PRV724 intravitreal injection. 1 × 10 9 pfu/mL PRV724 infected fewer RGCs than 3 × 10 9 pfu/mL PRV724. Confocal images of the retina in the control group after intravitreal injection of 1 × 10 9 pfu/mL ( n = 3 eyes) or 3 × 10 9 pfu/mL PRV724 (red) ( n = 3 eyes), co-stained with RBPMS (Alexa Fluor 488, green). (C) The contralateral retinas were not infected with PRV724. Confocal images of the retina contralateral to the PRV724-injected eye (red) ( n = 3 eyes), co-stained with RBPMS (Alexa Fluro 488, green). (D) An intravitreal injection of PBS produced no mRFP signals compared with an intravitreal injection of PRV724. Confocal images of retinas from PBS-injected eyes ( n = 3 eyes) co-stained with RBPMS (Alexa Fluro 488, green). (E) PRV724 did not infect fewer RGCs in the ONC mice than in the control mice. Confocal images of retinas from ONC mice with PRV724-injected eyes ( n = 3 eyes) co-stained with RBPMS (Alexa Fluro 488, green). (F) PRV724 infected a higher ratio of RGCs in the ONC group than in the control group. Co-labeling of RGCs and PRV724 was observed from flat-mounted retinas. Figure shows magnified confocal images of flat-mounted retinas from both control and ONC mice after an intravitreal injection of PRV724 (red) ( n = 3 eyes), with RGCs stained with RBPMS (Alexa Flour 488, green). Yellow triangles show the co-labeled RGCs (mRFP+ RGCs). (G) PRV724 infected a greater ratio of RGCs in the ONC group than in the control group. Co-labeling of RGCs and PRV724 was observed from cross-sectioned retinas. Figure shows retinal cross-sections from both control and ONC mice after an intravitreal injection of PRV724 (red) ( n = 3 eyes), with RGCs stained with RBPMS (Alexa Flour 488, green) and nuclei stained with DAPI (blue). Red arrows show the co-labeled RGCs. (H) Quantification of the RGCs in the control group and the PRV724-injected control group ( n = 3 eyes per group; the number of RGCs in the control group is shown in ). (I) Quantification of the RGCs in the ONC group and the PRV724-injected ONC group ( n = 3 eyes per group; the number of RGCs in the ONC group is shown in ). (J) Quantification of the ratio of mRFP+ RGCs/total RGCs in the control ( n = 16 eyes) and PRV724-injected ONC groups ( n = 11 eyes). (K) PRV724 did not infect fewer ipRGCs in the ONC group than in the control group. Confocal immunostaining images of cross-sections from wild-type mice in the control group and ONC group after an intravitreal injection of PRV724 (red). The ipRGCs were stained with opsin 4 (Alexa Flour 488, green) and the nuclei were stained with DAPI (blue) ( n = 3 eyes per group). Green triangles indicate ipRGCs. Red triangles indicate PRV724-labeled cells. Yellow triangles show the PRV724-infected ipRGCs. Scale bars: 50 µm. Data from 3 independent experiments are presented as the mean ± SEM. * P < 0.05 (two-way analysis of variance followed by Sidak’s multiple comparisons). DAPI: Diamidino-phenyl-indole; ipRGCs: intrinsically photosensitive retinal ganglion cells; mRFP: monomeric red fluorescent protein; ONC: optic nerve crush; PRV: pseudorabies virus; RBPMS: RNA binding protein with multiple splicing; RGCs: retinal ganglion cells.

Article Snippet: After three washes with PBS-0.1% Tween 20, goat anti-rabbit Alexa Fluor 488 (1:500, CST, Cat#4412S, RRID: AB_1904025) or goat anti-mouse Alexa Fluor 647 (1:500, CST, Cat#4410, RRID: AB_1904023) was applied.

Techniques: Labeling, Injection, Infection, Staining, Produced, Immunostaining, RNA Binding Assay

Journal: BMC Cancer

Article Title: Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

doi: 10.1186/s12885-023-10696-7

Figure Lengend Snippet: Antibodies used for Western blot (WB), immunocytochemistry (ICC) or immunohistochemistry (IHC) and immunofluorescence (IF).

Article Snippet: , Connexin 43 , FS1, TCam-2 , Rabbit polyclonal antibody, 3512, NEB * / Cell Signaling , 1:100 , Alexa Fluor 488 anti-Rabbit, A-11,008, Invitrogen, 1:5000.

Techniques: Western Blot, Immunocytochemistry, Immunohistochemistry, Immunofluorescence, Diagnostic Assay

Journal: Investigative Ophthalmology & Visual Science

Article Title: Proposed Mechanism of Long-Term Intraocular Pressure Lowering With the Bimatoprost Implant

doi: 10.1167/iovs.64.3.15

Figure Lengend Snippet: Treatment of human trabecular meshwork (TM) cells with implant levels of bimatoprost reduced fibronectin secretion and deposition. Panel ( A ) shows analysis of fibronectin in conditioned media from 12 different TM cell strains that were treated with bimatoprost (BIM; 1000 µM) or vehicle control (CON) for 24 hours. Panel ( B ) shows immunofluorescence images of fibronectin content in extracellular matrix of confluent TM cells (TM86) treated with or without bimatoprost for 24 hours. Fibronectin content was generally less complex and less abundant in treated samples, but was variable depending upon cell strain examined . The line and cross in the box whisker plots correspond to the median and mean, respectively. The top and bottom edges of the box indicate the 25th and 75th percentiles, and the whiskers extend to the most extreme data points. * P = 0.00005 versus CON.

Article Snippet: Cells on coverslips were then incubated overnight at 4°C with anti-fibronectin IgGs conjugated to Alexa Fluor 488 (Cell Signaling) in Cell Signaling Antibody dilution buffer (PBS with 1% BSA and 0.3% Triton X-100).

Techniques: Immunofluorescence, Whisker Assay