Plan apochromat 20x 0.8 objective

All images were taken on a lsm 700 confocal microscope (Zeiss) equipped with 405 nm (5 mW fiber output), 488 nm (10 mW fiber output), 555 nm (10 mW fiber output) and 639 nm (5 mW fiber output) diode lasers, a main dichroic beam splitter URGB and a gradient secondary beam splitter for lsm 700 using a 10x EC Plan-Neofluar (10x/0.3) or a 20x Plan-Apochromat (20x/0.8) objective (Zeiss). Image acquisition was done with ZEN 2010 (Zeiss), and image dimensions were 1024×1024 or 2048×2048 pixels with an image depth of 8, 12 or 16 bit. Two times averaging was applied during image acquisition. Laser power and gain were adjusted to avoid saturation of single pixels. Adjustment of brightness/contrast, changing of artificial colors (LUT), and maximum projections of Z-stack images were done in Fiji/Image J.

We performed the experiments after consolidation of the method papers ^{69 (link), 71}. Four to six week old cultures of human direct induced neurons were loaded with 10 μM FM_4–64 (Invitrogen) for 2 min in saline solution containing 170 mM NaCl, 3.5 mM KCl, 0.4 mM KH₂PO₄, 5 mM NaHCO₃, 1.2 mM Na₂SO₄, 1.2 mM MgCl₂, 1.3 mM CaCl₂, 5 mM glucose, 20 mM N-tris(hydroxymethyl)-methyl-2-aminoethane-sulfonic acid, pH 7.4) supplemented with 60 mM KCl. The cells were rinsed with saline solution only and then incubated with 10 μM FM_4–64 in just saline solution. The cells were washed three times with saline solution for a total of 5 min, followed by a wash for 1 min with 1 mM ADVASEP-7 (Sigma-Aldrich) in saline solution. FM imaging was performed at the LSM 710 confocal microscope (Zeiss) with a Plan-Apochromat 20X/0.8 objective with image taken every 5 s at 25°C. A one-minute baseline was recorded, followed by stimulation with 60 mM KCl in saline solution for 4 min. Cells were excited at 558 nm and the emission measured at 734 nm. Images were analyzed blinded in Fiji using the “plot z-axis profile” plug-in. We analyzed potential functional nerve terminals located along the GFP-positive cell (Supplementary Fig. 2). The FM_4–64 signal was determined by F = (F₁–B₁)/(F₀–B₀). Signal was normalized to mean fluorescence intensity measured at baseline condition.

Vibratome sections (100 μm thick) from virus-injected animals were immunolabeled with antibodies against mCherry, which was expressed from the virus, as well as antibodies against the mossy fiber marker, ZnT3. CellProfiler (Version 2.1.0) software was used to quantify the degree of overlap between mCherry and ZnT3. The average amount of overlap was 70%.
Primary antibodies: rat BrdU (AbD Serotec, MCA2060GA, 1:500), rabbit GFP (Invitrogen, A11122, 1:300), rabbit Complexin-1 (Proteintech, 10246–2–AP, 1:300), mouse GFAP (G6171, 1:500), mouse Nsf-1 (MA1–12435, 1:300) and rabbit Syn3 (OSS00018W, 1:300) from Thermo Scientific, guinea pig Iba1 (234 004, 1:500), mouse Syt1 (105 011, 1:300), guinea pig ZnT3 (197 004, 1:1000) from Synaptic Systems.
Secondary antibodies. Antibodies (all obtained from Jackson Laboratory, donkey anti-guinea pig IgG (H+L) ML, #706-225-148 and 706-175-148; donkey anti-mouse IgG (H+L) ML, #715-225-151, 715-165-151, and 715-175-151; donkey anti-rabbit IgG (H+L) ML, #711-225-152, 711-165-152, and 711-175-152 were diluted 1:400. Confocal scans were carried out using a LSM 510 Imager Z.1 microscope (Zeiss) using Plan-Apochromat 63X/1.4 and EC Plan-Neofluar 40X/1.30 oil immersion objective lenses and four excitation laser lines. Tile scans were performed using a LSM 710 confocal microscope (Zeiss) with a Plan-Apochromat 20X/0.8 objective.

Eyes from Flk1-H2B::YFP^+/tg mice (E12.5 to P10) were enucleated and immediately placed on a 35 mm glass bottom petri dish (MatTek) with the corneal surface facing the glass. The eyes were then imaged en face using a Zeiss LSM 510 META confocal microscope and Plan-Apochromat 20X/0.8 objective. Using LSM Image Examiner software and either the rim of the ciliary body in the embryos or the postnatal iris as a guide, the PM within each image was cropped and its area measured. This was followed by manual counting of every Flk1-H2B::YFP+ cell within the entire PM. Statistically significant differences (t-test) between varying time points were determined for the total number of cells per PM and the number of cells per PM area.

Islet cell viability of mouse pancreatic islets and pseudo-islets was examined upon 1.5 mM STZ treatment for 24 h, using the LIVE-DEAD Viability-Cytotoxicity Kit (Thermo Fisher Scientific). Following treatment, whole islets were incubated protected from light for 1 h at 37°C and 5% CO₂ with 10 μg/mL Hoechst 33342 (DNA stain), 2 μM Calcein AM (live cells), and 4 μM Ethidium homodimer-1 (EthD-1, dead cells) in KRH buffer supplemented with 0.1% BSA (Sigma-Aldrich) and 10 mM glucose (Sigma-Aldrich) to co-stain cell nuclei, as well as viable and dead cells. Afterward, Z-stack images of the stained islets were acquired using a Zeiss LSM 710 coupled to an Axio Observer.Z1 microscope (Carl Zeiss MicroImaging GmbH) equipped with a Plan-Apochromat 20x/0.8 objective or Zeiss ApoTome (Carl Zeiss MicroImaging GmbH) equipped with a Plan-Apochromat 103/0.45 objective. All images were analyzed using Fiji (ImageJ) image analysis software and cell death was quantified by using the semi-automated quantification macro as previously published by Scholz and colleagues.^{64 (link)} The area of dead (EthD-1-positive) cells of each islet was normalized to the whole islet cell area (Hoechst-positive).