Journal: iScience

Article Title: Transplanted human induced pluripotent stem cells- derived retinal ganglion cells embed within mouse retinas and are electrophysiologically functional

doi: 10.1016/j.isci.2022.105308

Figure Lengend Snippet: Immuno-staining of the transplanted hiPSC-RGCs in mouse retina with RGC-specific markers (A–H) Flattened whole-mount images of transplanted hiPSC-RGCs ( n = 8) were co-stained with RGC-specific markers (A) BRN3, (B) RBPMS, (C) TUJ1, (D) MAP2, (E) VGLUT2, and (F) PSD95. Donor hiPSC-RGCs were also co-stained with (G) human nuclear antigen (HNA) and (H) Ku80 to confirm the donor origin of SNCG-eGFP + cells. ∗ Since the human nuclear antigen antibody was raised in mice, some cross-reactivity with the murine retina was observed. Z-1: Zoom-1 (scale bar is 10 μm), Z-2: Zoom-2 (scale bar is 10 μm), and Z-4: Zoom-4 (scale bar is 5 μm).

Article Snippet: Retinas were prepared as flattened whole mounts ( n = 8), washed with 1X PBS three times, and permeabilized in 0.5% Triton X-100 in PBS by freezing at −80°C for 15 min. Retinas were incubated overnight at 4°C in a humidified chamber with either BRN3 (Santa Cruz, catalog #: sc-6026, Dallas, TX, USA), RBPMS (Sigma-Aldrich, catalog #: ABN1362, St. Louis, MO, USA), Tubulin β 3 (TUJ1; BioLegend, catalog #: 801201, San Diego, CA, USA), GFP (Novus Biologicals, catalog #: NB600-308, Littleton, CO, USA), PSD95 (Cell Signaling Technology, catalog #: 3409S, Danvers, MA, USA), VGLUT2 (Cell Signaling Technology, catalog #: 14487S, Danvers, MA, USA), Ku80 (Cell Signaling Technology, catalog #: 2180S, Danvers, MA, USA), or Human Nuclear Antigen (Novus Biologicals, catalog #: NBP2-34342, Littleton, CO, USA).

Techniques: Immunostaining, Staining

Journal: iScience

Article Title: Transplanted human induced pluripotent stem cells- derived retinal ganglion cells embed within mouse retinas and are electrophysiologically functional

doi: 10.1016/j.isci.2022.105308

Figure Lengend Snippet:

Article Snippet: Retinas were prepared as flattened whole mounts ( n = 8), washed with 1X PBS three times, and permeabilized in 0.5% Triton X-100 in PBS by freezing at −80°C for 15 min. Retinas were incubated overnight at 4°C in a humidified chamber with either BRN3 (Santa Cruz, catalog #: sc-6026, Dallas, TX, USA), RBPMS (Sigma-Aldrich, catalog #: ABN1362, St. Louis, MO, USA), Tubulin β 3 (TUJ1; BioLegend, catalog #: 801201, San Diego, CA, USA), GFP (Novus Biologicals, catalog #: NB600-308, Littleton, CO, USA), PSD95 (Cell Signaling Technology, catalog #: 3409S, Danvers, MA, USA), VGLUT2 (Cell Signaling Technology, catalog #: 14487S, Danvers, MA, USA), Ku80 (Cell Signaling Technology, catalog #: 2180S, Danvers, MA, USA), or Human Nuclear Antigen (Novus Biologicals, catalog #: NBP2-34342, Littleton, CO, USA).

Techniques: Virus, Recombinant, RNA Sequencing, Software

Journal: bioRxiv

Article Title: Optogenetic stimulation of medial septal glutamatergic neurons modulates theta-gamma coupling in the hippocampus

doi: 10.1101/2022.03.15.484394

Figure Lengend Snippet: A) Immunofluorescence images from brain slices show co-expression of ChR2-YFP (green) and VGLUT2 in the medial septal area of a transgenic mouse. B)Left-LFP traces and single responses to light stimulation in MSA, CA1 and CA3, note time delay between depolarization of MSA and hippocampus. Right – Min-Max boxplots showing time delay between CA3 and CA1 to first 50 light stimuli. C. Raw (black) and filtered (blue - slow gamma band, red – fast gamma) records of hippocampal LFP during optical stimulation of MSA at 5 and 8 Hz. D) Power spectral density (PSD) graphs of LFPs from CA1 before(black), during(red) and after (blue) optical stimulation of different frequencies. E) Min-Max boxplots showing integrated (PSD) depending on the frequency of stimulation for theta (top), slow gamma (middle) and fast gamma (bottom) bands.

Article Snippet: After incubation, the sections were washed with a buffer and antibodies to VGluT2 + Alexa Fluor 647 (Novus Biologicals) were added to them for 2 hours.

Techniques: Immunofluorescence, Expressing, Transgenic Assay

Journal: Journal of Neuroscience

Article Title: Reconstitution of Giant Mammalian Synapses in Culture for Molecular Functional and Imaging Studies

doi: 10.1523/jneurosci.3869-15.2016

Figure Lengend Snippet: Figure 5. shRNA knockdown of presynaptic proteins in calyceal terminals in culture. Cultured cells were infected at DIV8 with shRNA lentiviral particles specific for VGLUT2. Scrambled shRNA lentiviralparticleswereusedasanegativecontrol.ExperimentswereperformedatDIV18.a,VGLUT1(toppanels;green)andVGLUT2(bottompanels;red)immunofluorescenceimagesofcalyceal terminalsinfectedwitheitherscrambledshRNA(leftpanels)orVGLUT2-specificshRNA(rightpanels).Scalebar,10m.BargraphsrepresentimmunofluorescencesignalintensitiesofVGLUT1(top) andVGLUT2(bottom)afterinfectionwithscrambled(blackbars)orVGLUT2-specific(redbars)shRNAs.Apparentknockdownefficiencywas55%forVGLUT2(n 11each,p 0.027comparedwith scrambledshRNA;fordetails,seeMaterialsandMethods).b,EvokedEPSCsatcalycealsynapsesincultureinfectedwithscrambled(black)orVGLUT2-specific(red)shRNAs.Bargraphsrepresentmean EPSCamplitudes(1092286pAinscrambledshRNAcontrolvs31161pAinVGLUT2-shRNA;n 5andn 6,p 0.017).Bottomleft,BargraphsrepresentthePPRatinterpulseintervalsof 20 ms. Bottom right, Bar graphs represent the coefficient of variation (CV SD/mean) of the EPSC amplitude. Bar graphs are color-coded as EPSC traces. *p 0.05.

Article Snippet: Lentiviral-mediated shRNA knockdown of VGLUT2 was accomplished with commercially available lentiviral particles (sc-42333-V; Santa Cruz Biotechnology); 3.3 10 5 units of lentiviral particles was added to one 35 mm dish of culture at DIV8.

Techniques: shRNA, Knockdown, Cell Culture, Infection

Journal: iScience

Article Title: Novel human pluripotent stem cell-derived hypothalamus organoids demonstrate cellular diversity

doi: 10.1016/j.isci.2023.107525

Figure Lengend Snippet:

Article Snippet: Mouse Monoclonal Anti-VGLUT2 (Clone CL2921) , Atlas Antibodies , Cat#AMAb91081; RRID: AB_2665793.

Techniques: Recombinant, Membrane, Protease Inhibitor, Reverse Transcription, Bicinchoninic Acid Protein Assay, SYBR Green Assay, Lysis, Extraction, Western Blot, Software, Fluorescence, Sterility, Real-time Polymerase Chain Reaction