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 , 1:1000 , Goat anti-rabbit IgG-HRP, sc-2004, Santa Cruz, 1:5000.

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

Journal: Molecular Pain

Article Title: Inhibition of NKCC1 in spinal dorsal horn and dorsal root ganglion results in alleviation of neuropathic pain in rats with spinal cord contusion

doi: 10.1177/17448069231159855

Figure Lengend Snippet: Inhibition of NKCC1 in DRGs. (a): The mRNA expression level of NKCC1 gene was detected by qRT-PCR. (b): The protein level of NKCC1 was detected by WB analysis. (c): NKCC1 protein concentration in the three groups using the BCA assay. (d): DAPI staining of DRGs section with outlined spinal nerve dorsal root area and a squared inset of observed ganglion. (e-f): Quantification of NKCC1 immunoreactivity (green) with representative immunofluorescence images of DRGs sections. DAPI, blue; scale bar, 50 μm.

Article Snippet: Slides were blocked with normal goat serum for 30 min at 37°C, incubated with antibody against NKCC1 (#85403, CST, USA) overnight at 4°C, rinsed with 0.01 M phosphate buffered saline (PBS) 3 min for three times each, incubated with FITC-(for NKCC1) conjugated goat anti-rabbit IgG antibodies for 30 min at 37°C, and co-stained with DAPI.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Protein Concentration, BIA-KA, Staining, Immunofluorescence

Journal: Molecular Pain

Article Title: Inhibition of NKCC1 in spinal dorsal horn and dorsal root ganglion results in alleviation of neuropathic pain in rats with spinal cord contusion

doi: 10.1177/17448069231159855

Figure Lengend Snippet: Inhibition of NKCC1 in spinal cords. (a): The mRNA expression level of NKCC1 gene was detected by qRT-PCR. (b): The protein level of NKCC1 was detected by WB analysis. (c): NKCC1 protein concentration in the three groups using the BCA assay. (d): DAPI staining of spinal cord section with outlined gray matter area and a squared inset of observed ventral horn. (e): Immunostaining of NKCC1 (green) in the T10 region of spinal cords in different groups. DAPI, blue; sale bar, 50 μm. (f): Quantification of NKCC1 immunoreactivity. n.s.: Not significant between the three groups.

Article Snippet: Slides were blocked with normal goat serum for 30 min at 37°C, incubated with antibody against NKCC1 (#85403, CST, USA) overnight at 4°C, rinsed with 0.01 M phosphate buffered saline (PBS) 3 min for three times each, incubated with FITC-(for NKCC1) conjugated goat anti-rabbit IgG antibodies for 30 min at 37°C, and co-stained with DAPI.

Techniques: Inhibition, Expressing, Quantitative RT-PCR, Protein Concentration, BIA-KA, Staining, Immunostaining