Lsm 700 confocal fluorescence microscope

After the irradiation of 2 Gy using X-RAD 320 (Precision X-Ray, North Branford, CT, USA), 2000 cells were plated on 4-well slide plates. After plating, the cells were treated with PI3K kinase inhibitors (GDC0941, GDC0032, CAL101 and IPI145) for 12 hours. The cells were fixed by a 4% paraformaldehyde solution. The cells were permeabilized by incubating in phosphate-buffered saline (PBS) containing 0.25% Triton X-100 for 10 min followed by washing three times with PBS for 5 min. Next, the cells were blocked with 1% BSA in PBS with 0.1% Tween 20 (PBST) for 1 h. The cells were incubated overnight at 4°C with anti-phospho-γ-H2AX antibody (05636I, Millipore, Burlington, Massachusetts, USA), and washed three times for 5 minutes with PBST. The cells were incubated with a fluorescein isothiocyanate-labeled secondary antibody for 1 h in the dark and mounted with VECTASHIELD solution (94 010, Vector Laboratories, Burlingame USA). Observation phospho-γ-H2AX foci were observed and fluorescence images were acquired using the Zeiss LSM 700 confocal fluorescence microscope.

Eyes were dissected and immunostained in flat mounts as described previously (20 (link)). Briefly, dissected eyes were fixed in 4% PFA overnight at 4°C and carefully dissected. Nonspecific binding was prevented by blocking with Dako’s serum-free blocking buffer, and all specimens were incubated with anti-Brn3a antibody (1:200; AB5945, MilliporeSigma) at 4°C for 5 nights. After incubation with Alexa Fluor 568–conjugated rabbit anti–rabbit IgG (1:1,000; Life Technologies, Thermo Fisher Scientific), retinas were mounted in Ultramount Aqueous Permanent Mounting Medium (DakoCytomation). Four micrographs (700 × 700 μm) per single retinal specimen from the mid-peripheral region of quadrants 1.5–2.0 mm from the optic head were imaged using an LSM 700 confocal fluorescence microscope (Zeiss). Brn3a⁺ RGCs were counted using ImageJ (NIH) and averaged for at least 3 retinas per group.

The sections were prepared as described above. For immunohistochemistry, sections were deparaffinized in xylene and dehydrated through a graded series of alcohol. High-pressure antigen retrieval was performed with citrate antigen repair solution and then incubated in 3% hydrogen peroxide at room temperature for 20 min. The slices were incubated with primary rabbit polyclonal antibodies against stromal cell-derived factor-1 (SDF-1, 1:200 dilution, Abcam, Cambridge, MA) at 4 °C overnight and then incubated with a horseradish peroxidase-labeled secondary antibody at 37 °C for 30 min. Next, 3,3′-diaminobenzidine (DAB) was added at room temperature for 10 min, and the slices were then stained with hematoxylin at room temperature for 2 min. Finally, the slices were gently washed with deionized water, dehydrated in gradient alcohol solutions, mounted with neutral balsam, and observed using an optical microscope. Anti-CD31 and anti-CD34 antibodies (1:200 dilution, Abcam, Cambridge, MA) were used for immunofluorescence. As for immunofluorescence, the slices were incubated with the abovementioned primary antibodies at 4 °C overnight and then incubated with a secondary antibody at 37 °C for 1 h. The slices were finally stained with 4′,6-diamidino-2-phenylindole (DAPI, Invitrogen, Carlsbad, CA) and observed under a Zeiss LSM 700 confocal fluorescence microscope (ZEISS, Germany).

Actin cytoskeletons, GA, and nuclei were visualized using immunofluorescence staining subsequent to 3 h or pulsed ES. The electrically treated cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then washed twice with Dulbecco’s phosphate-buffered saline (DPBS). Cells were permeabilized with 0.1% Triton X-100 in DPBS for 5 min at RT and then washed three times with DPBS for 5 min. Bovine serum albumin (1%; BSA) was added and left to incubate for 30 min at RT to block nonspecific binding. The cells were then incubated with a GM130 primary antibody (dilution 1:250; Abcam, Cambridge, UK) and treated with Alexa Fluor^® 488 phalloidin (5U/mL; A12379, Invitrogen, Carlsbad, CA, USA), at 4 °C overnight to label the actin cytoskeleton. Cells were then rinsed at least three times with DPBS and treated with goat anti-Rabbit IgG conjugated with Texas Red (dilution 1:1000; T-2767, Invitrogen) for 1 h at RT in the dark to label GM130. After DPBS washing, cells were treated with Hoechst #33258 (B2883, Sigma-Aldrich, St. Louis, MO, USA) for 10 min at RT in the dark before being rinsed twice with DPBS. Fluorescence images were acquired using an LSM700 confocal fluorescence microscope (Carl Zeiss, White Plains, NY, USA).

Cultures of MG1655 araE_CP containing the plasmid pSEB293 (Pichoff and Lutkenhaus, 2005 (link)) encoding GFP-FtsA, Gfp-FtsA(E14R), Gfp-FtsA(S84L), Gfp-FtsA(A188V), Gfp-FtsA(D210A), and Gfp-FtsA(Y375A) were grown overnight on solid LB Lennox media containing ampicillin (100 μg ml^–1) without arabinose (leaky expression was sufficient to observe localization) at 30°C and then streaked onto plates of the same composition and grown for 5 h at 30°C. Cells were collected and resuspended in 1x phosphate buffered saline (PBS) and, where indicated, incubated for 10 min in the dark at room temperature with FM 4−64FX (3 μg ml^–1) (ThermoFisher). Cells were applied to a 5% agarose pad containing M9 minimal media with glucose (0.4%), and then a coverslip was added. Samples were visualized with a Zeiss LSM 700 confocal fluorescence microscope with excitation at 488 nm and emission at 555 nm for Gfp and 565 nm and 744 nm for FM 4−64FX, respectively. Where indicated, a Nomarski prism was used to acquire differential interference contrast (DIC) images. All images were captured on an AxioCam digital camera with ZEN 2012 software. Cell lengths were measured using NIH ImageJ.

The imaging of cells was conducted using the Zeiss LSM-700 confocal fluorescence microscope and observer D1 fluorescence microscope with Zeiss AxioCam MRC camera. The images were processed by ZEN software.
The thickness of BrM was measured using transmission microscopy. The mice were perfused with 2.5% glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) and prepared with microscope system laboratory at UNC for post-fixation, dehydration and embedding in epoxy resin. Toluidine blue was stained at semi-thin sections (1 um), and lead citrate for ultrathin sections. BrM thickness was measured using JEOL 1230 TEM at 15000× magnification, and 10 random measurements were averaged in each sample (3 samples per group).