Immunocytochemistry and Microscopy of 2D-Cultured Cells, Whole-Mount Spheroid Preparations, and Cryosections

2D-cultured cells fibronectin-coated polystyrene culture dishes (Nunc) were washed with PBS then fixed with 3% para-formaldehyde in PBS for 15 min, permeabilized with 0.2% Triton-X100 (Sigma) in PBS for 10 min, incubated for 30 min with bovine serum albumin (Sigma) 1 mg/mL in PBS at room temperature, incubated over night with primary antibodies at 4°C, washed three times with PBS and incubated 1 h with secondary antibodies goat anti mouse or anti rabbit coupled to Alexa fluorescent dyes (Invitrogen). DAPI (Sigma) was added to secondary antibodies to visualize the nuclei or Vectashield mounting medium with DAPI was used (Vectorlabs). Preparations were examined on a Zeiss LSM 710 confocal microscope using 40× and 63× Zeiss oil immersion lenses (Carl Zeiss). For whole-mount immunostaining of entire spheroids, live spheroids either were allowed to adhere overnight onto fibronectin-coated glass-bottom dishes (MatTek) or 10–20 spheroids were collected by sedimentation and further incubation and washing steps were executed in 500 μL Eppendorf-tubes. After fixation with 3% para-formaldehyde (PFA) for 1 h in the cold, permeabilization and antibody incubation steps in 1% BSA/PBS/10% Tween-20 (Sigma) were prolonged to 1 day each in the cold before examination by confocal microscopy using a 20× air lens. For cryosections, spheroids were collected by gentle spinning in a tube. The spheroids were then fixed with 3% para-formaldehyde in PBS for 60 min, washed with PBS, stored and embedded in OCT compound. Blocks were cut using a Zeiss Hyrax cryostat. Frozen sections on slides were air dried for at least 1 h and then post-fixed in cold acetone. After a blocking step with 1% BSA/PBS, primary antibodies in 1% BSA/PBS, and 0.3% Tween20 were applied overnight. Further processing was the same as with 2D cultures. A sample of left ventricular, normal adult mouse heart was a gift from Maria Essers (Institute for Biochemistry and Molecular Medicine, Bern University) that was fixed with PFA, and used for cryosections and immunostaining as detailed above for 2D cultured cells.