Hc pl apo 20x 0.75 imm

For confocal imaging, fibroblasts were cultured on CyPhyGels of different stiffness for 4 days, fixed using 4% paraformaldehyde (Roth, Germany) in PBS for 15 min. Glycoproteins at the cell membrane were stained with AlexaFluor555-conjugated wheat germ agglutinin (WGA, 1 μg/mL, Invitrogen, Germany) in 3% bovine serum albumin in PBS for 1 h, followed by three 10-min washing steps in PBS. Thereafter, cells were permeabilized using 0.5% Triton-X100 (Sigma-Aldrich, Germany) in PBS for 15 min and blocked using 3% bovine serum albumin in PBS for 1 h. Primary antibody against alpha smooth muscle actin (αSMA; ab7817, Abcam, Germany) was applied 1:50 in 1% bovine serum albumin in PBS overnight. After three washing steps in PBS, AlexaFluor488-coupled secondary anti-mouse was applied 1:500 in PBS for 1 h. Nuclear counterstaining was performed using Hoechst-33342 (20 μM, ThermoFisher, Germany) for 2–3 min in PBS. All staining was performed at room temperature.
Imaging was performed on a Leica TCS SP8 X confocal microscope using a 20× multi-immersion objective (HC PL APO 20x/0.75 IMM) with water as immersion medium. For imaging, the CyPhyGels were mounted upside-down in a PBS-filled microscopy dish (µ-Dish 35 mm, ibidi, Germany). Cell area was determined using ImageJ after manually tracing cell borders based on WGA signal.