35 mm glass bottom dishes

Untreated and AGI5198 treated cells (U251^WT, U251^R132H, and U251^R132C) were seeded in the luminescence free 35 mm glass bottom dishes (Fisher Scientific Co, Hanover Park IL) using media described above. Right before the experiments, media was replaced with the warmed optically transparent DMEM media (Life Technologies Corporation, Grand Island NY) and dishes were covered with a 22 mm circle cover glasses (VWR Scientific, Chicago, IL) and sealing with a waterproof silicon sealant. To determine organelle-specific Raman spectra, we labeled each type of organelle, one at a time, with the following markers, MitoTracker Green FM (Life Technologies Corporation, Grand Island NY) for mitochondria, ER-Tracker Green (Life Technologies Corporation, Grand Island NY), for Endoplasmic Reticulum, and NBD C6 ceramide-BSA (Life Technologies Corporation, Grand Island NY) for Golgi Apparatus, following the manufacturer’s instructions. At the end of labeling, the cells were washed with warmed, sterile PBS three times and then the optically transparent DMEM supplemented with 25 mM of HEPES was added. Optimal labeling time was confirmed via confocal microscopy^{25 (link)}.

For fixed cell colocalization experiments, cells seeded on 13 mm coverslips (placed in 24-well plates) were transfected using Lipofectamine 2000 (Thermo Fisher) or Nanofectin (PAA) and 10–500 ng DNA depending on the plasmids and the experiments (low or high overexpression). The levels of each plasmid were titrated down to low levels allowing good detection but limiting side effects of overexpression. Cells seeded onto live-cell imaging 35 mm glass bottom dishes (MatTek) were transfected using Lipofectamine 2000 (Thermo Fisher) and 50–250 ng DNA. For pull-down experiments, co-immunoprecipitation and EGFP-trap immunopurifications, HEK293 cells seeded in 6-well plates or 100 mm dishes were transfected using GeneJuice (Merck) and 1–3 μg DNA. Cells were incubated 16–24 h to express the constructs and were either imaged live, fixed (4% pre-warmed paraformaldehyde, 20 min at 37 °C) or processed to prepare cell extracts.

After culturing, the intestinal organoids were planted on 35-mm glass bottom dishes (Thermo Fisher Scientific, Cat#150682) and fixed by 4% cold paraformaldehyde, washed with cold PBS and blocked in Blocking buffer (1× PBS, 5% anti-goat serum, 0.01% Triton X-100) for 1 h. Then the fixed organoids were successively incubated with anti-DDDDK tag primary antibodies (Abcam, Cat#ab1162) or anti-Ki67 primary antibodies (Abcam, Cat#ab15580) at 4 °C overnight and secondary antibodies (Abcam, Cat#ab150073) or (Abcam, Cat#ab150062) at room temperature for 90 min. For frozen-section staining, optimal cutting temperature compound (Leica, Cat#14020108926) embedded intestinal cryosections of 10–14 μm thickness were stained with the DAPI for 5 min at room temperature. The confocal images were acquired using an Olympus FV1200 confocal microscope (Japan).

Confocal microscopy was performed on ObaGel- and silk-based in vitro adipogenic differentiated constructs (see Adipogenic Differentiation) and explants following 6-week in vivo implantation and staining with BODIPY lipophilic dye. Confocal research was conducted using a Nikon A1 LASER Ti-E microscope system. The tissue was imaged in Nunc 35 mm glass bottom dishes, at 10x and 20x oil-free objectives, using the FITC and DAPI LASERs. Cryoscanning electron microscopy was conducted on ObaGel constructs and silk scaffolds that were seeded with human SVF cells following a 2-week adipogenic differentiation. Cryo-SEM structure research was done with Gatan 2500 alto Cryo-system and Hitachi S-4800 SEM. The tissue was cut to 7 × 5 × 5 (mm) and laid about 5 mm high above a cryogenic SEM sample holder surface, clamped and glued into the holder, and then frozen in slushed liquid nitrogen at approximately -210°C for about 30 seconds. The frozen tissue was transferred in vacuum to the prechamber attached to the SEM and then fractured and sublimed for 5 minutes at -95°C. After coating for 88 seconds at -130°C with Pt/Pd, the sample was moved to the SEM and observed at 3 kV at -130°C.

Imaging was performed as previously described^{58 (link)}. The testes were dissected in 1X Becker Ringer’s solution^{58 (link)} and then mounted onto a 35 mm Glass Bottom Dishes (Nunc). 500 μL of 1 mg/mL poly-L-lysine (Sigma) was pipetted onto the coverslip portion of the imaging dish and incubated for 5- to 7-hours at room temperature. Then, poly-L-lysine solution was replaced to the Becker Ringer’s solution and testes were mounted onto poly-L-lysine layer with the tip of the testes oriented toward bottom. Next, Becker Ringer’s solution was slowly removed and replaced with 3 ml of room temperature Schneider’s Drosophila medium supplied with 10% fetal bovine serum and glutamine–penicillin–streptomycin (Sigma).
Z-stacks (2 µm interval, for 11 stacks) were taken using Zeiss LSM800 airyscan, 1AU-pinhole with 63X oil immersion objective (NA = 1.4) every 10 minutes for overnight (16 hours). Preset tiling function (Zen software, Zeiss) was used for sequential imaging of multiple positions to obtain time-lapse images from 5-to-8 testes per night.