Journal: Neural Regeneration Research

Article Title: L- and T-type Ca 2+ channels dichotomously contribute to retinal ganglion cell injury in experimental glaucoma

doi: 10.4103/1673-5374.360277

Figure Lengend Snippet: Changes in protein levels of L- and T-type Ca 2+ channel subunits in COH retinas. (A) Representative immunoblots showing the expressions of Ca V 1.2 (a L-type Ca 2+ channel subunit) and Ca V 3.1, Ca v 3.2, and Ca V 3.3 (T-type Ca 2+ channel subunits) in sham-operated (control) and COH retinal extracts at the indicated post-operational times. (B–E) Bar charts showing the densitometric quantification of immunoreactive bands of Ca v 1.2 (B), Ca v 3.1 (C), CaV3.2 (D), and Ca V 3.3 (E) under the conditions as shown in A. (F) Representative immunoblots showing the expression of Brn3a, a RGC marker, in control and COH retinal extracts at the indicated post-operational times. (G, H) Bar charts showing Brn3a protein levels (G) and Ca v 1.2/Brn3a ratios (H) under the conditions as shown in A and F. Data are presented as the mean ± SEM ( n = 6 for all groups). * P < 0.05, ** P < 0.01, *** P < 0.001, vs . control (one-way analysis of variance test with Bonferroni’s post hoc test (for B–D, G and H) and non-parametric Kruskal-Wallis test (for E)). COH: Chronic ocular hypertension; RGC: retinal ganglion cell.

Article Snippet: The primary antibodies used in this study were mouse anti-Ca V 1.2 monoclonal antibody (1:50, Abcam, Cambridge, MA, USA, Cat# ab84814, RRID: AB_1860052), rabbit anti-Ca V 3.1 polyclonal antibody (1:100, Alomone, Jerusalem, Israel, Cat# ACC-021, RRID: AB_2039779), rabbit anti-Ca V 3.2 polyclonal antibody (1:100, Alomone, Cat# ACC-025, RRID: AB_2039781), rabbit anti-Ca V 3.3 polyclonal antibody (1:100, Alomone, Cat# ACC-009, RRID: AB_2039783), goat anti-Brn-3a polyclonal antibody (1:300, Santa Cruz Biotechnology, Santa Cruz, TX, USA, Cat# sc-31984, RRID: AB_2167511), and mouse anti-β-actin (1:10,000, Sigma-Aldrich, Cat# A5316, RRID: AB_476743).

Techniques: Western Blot, Control, Expressing, Marker, Analysis

Journal: Cell Reports

Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

doi: 10.1016/j.celrep.2020.107968

Figure Lengend Snippet: Multiplexed Immunolabeling in Epoxy-Resin-Embedded Tissue after Etching and Antigen Retrieval (A) Multiple immunolabeling of an epoxy-resin-embedded section of the hippocampal CA1 area of an adult rat. Four rounds of triple labeling were performed using primary Abs raised in 3 different species (top row: mouse monoclonal IgG1; middle: rabbit polyclonal; bottom: Guinea pig polyclonal Abs). Reactions within one round were visualized with Alexa488-, Cy3-, and Cy5-coupled sAbs (all reactions are pseudocolored and shown in cyan). Boxed areas are shown at higher magnifications in panels (B 1 and B 2 ). (B 1 ) Immunolabeling for PV, Kv2.1, VGAT, and vGluT1 are present in different, nonoverlapping subcellular compartments. The vGluT1-positive terminals are only present around the PV+ IN soma but not the neighboring PC somata that are outlined by the Kv2.1 labeling. VGAT-positive terminals (cyan) surround the somata of the IN and the PCs. (B 2 ) Immunolabeling of PV, PSD95, CB1, and neuroligin-2 (NL-2). (C) Confocal images of a 200-nm-thick section of the mouse hilar region labeled for PSD95 (1 st and 3 rd rounds) and for VGAT (2 nd and 4 th rounds; both Guinea pig primary Abs). Circular ROIs were placed over PSD95-labeled glutamatergic synapses (yellow) and over VGAT-positive axons (cyan). The nonspecific background labeling (white ROI) was measured over the unlabeled neuropil. (D) Mean normalized integrated fluorescence values are plotted for 4 labeling rounds. Open symbols represent reactions from 3 mice, and the filled symbols are mean ± SD. The fluorescence in the inhibitory axons is 2.6% ± 2.4% in the 1 st and 4.6% ± 2.5% in the 3 rd rounds, similar to that measured over the excitatory synapses in the 2 nd (7.2% ± 1.1%) and 4 th (5.7% ± 2.9%) rounds. A total of 21–28 ROIs were analyzed in each condition. str. rad., stratum radiatum; str. pyr., stratum pyramidale.

Article Snippet: Mouse monoclonal IgG1 anti-Cav3.1 , NeuroMab , Cat#75-206; RRID: AB_2069421.

Techniques: Immunolabeling, Labeling, Fluorescence

Journal: Cell Reports

Article Title: A High-Resolution Method for Quantitative Molecular Analysis of Functionally Characterized Individual Synapses

doi: 10.1016/j.celrep.2020.107968

Figure Lengend Snippet:

Article Snippet: Mouse monoclonal IgG1 anti-Cav3.1 , NeuroMab , Cat#75-206; RRID: AB_2069421.

Techniques: Virus, Plasmid Preparation, Recombinant, Software, Microscopy