Csu w1 spinning disc module

The filamentous actin (F-actin) in the cell cytoskeleton was stained using Alexa Fluor™ 594 Phalloidin (Thermo-Fisher, Waltham, MA, USA, Cat. No. A12381, 200 U/mL) and cell nuclei using DAPI (Thermo-Fisher, D1306, 300 nM concentration). Staining was performed in humidified chambers for 120 min at room temperature. After staining, the samples were rinsed and stored in PBS. Confocal microscopic images were taken using a Nikon CSU-W1 inverted spinning disc confocal microscope based on the Nikon Eclipse Ti2 inverted microscope (Nikon, Tokyo, Japan) with the Yokogawa CSU-W1 spinning disc module (Yokogawa, Tokyo, Japan) and equipped with dual sCMOS PRIME BSI cameras (Teledyne Photometrics, Tucson, AZ, USA). Due to the manner of the samples, Nikon dry objectives CFI Plan Apo VC 20x were used with a 50 mm pinhole disc in Z stack mode (–0–250 mm depth). Images were then processed in Imaris 10.1.0 software (Oxford Instruments, Abingdon-on-Thames, UK).

Filamentous actin (F-actin) was visualized in the cell cytoskeleton using Alexa Fluor^TM 594 phalloidin (Thermo-Fisher, Waltham, MA, USA, cat. no. A12381, 200 U/mL) and cell nuclei using DAPI (Thermo-Fisher, D1306, concentration 300 nM). As an early marker of SMC differentiation, we used Alexa Fluor™ 488 (Thermo-Fisher, Waltham, MA, USA, concentration 2 μg/mL). The samples were fixated using Palay solution for approximately one hour, then permeabilized using 0.1% Triton-X 100 in PBS for 10 min and blocked using blocking solution (1% BSA, 22.52 mg/mL glycine in PBST (PBS + 0.1% Tween 20)) for 25 min. Staining was performed in humidified chambers at room temperature for 120 min, after which the samples were rinsed several times and stored in PBS.
Confocal microscopy was performed using a Nikon CSU-W1 inverted spinning disk microscope, which was based on a Nikon Eclipse Ti2 inverted microscope (Nikon, Tokyo, Japan) with the Yokogawa CSU-W1 spinning disc module (Yokogawa, Tokyo, Japan) and equipped with dual sCMOS PRIME BSI cameras (Teledyne Photometrics, Tucson, AZ, USA). Nikon CFI Plan Apo VC 20× dry lenses with a 25 mm pinhole disc were used in Z stack mode (depth −0–250 mm) to image the samples. The images were captured and subsequently processed using the Imaris 10.1.0 software (Oxford Instruments, Abingdon-on-Thames, UK).