Briefly, muscles were removed immediately after sacrifice and pinned to Sylgard-coated dishes for fixation with 2% paraformaldehyde in 0.1 M phosphate buffer for 1 h, followed by fixation in methanol (−20°C) for 6 min. Single fibers were obtained by manual teasing.
Fig. 8a shows a tipical, grey-scale, DH microscope phase-image of living-cells in a Petri dish without any fixation process.
For the alkaline phosphatase staining cells were cultured on dishes, followed by fixation and stained according to the manufactures protocol (Sigma).
After fixation, the dishes, membranes and tissue were washed with phosphate buffer, cut out and kept in phosphate buffer.
After fixation, the Petri dish was stained with PE-labeled anti-CD41 mAb (DAKO) and viewed with a fluorescent microscope.
Various methods have been described in order to support the capsular bag during culture: external fixation of the capsule to the culture dish using glue [ 13] or entomological pins [ 10, 14], the fixation of the ciliary body [ 11], or internal capsule expansion using Capsular Tension Rings (CTRs) [ 12].
After fixation of all dishes, immunofluorescence was performed as described.
Cells were trypsinized and plated onto the microwell dishes 24 hours prior to fixation and staining.
The cells from ∼80% confluent 10 cm dishes were crosslinked by adding fixation solution (1% formaldehyde, 0.1M NaCl, 1 mM EDTA, 50 mM HEPES⋅KOH pH 7.6) for 10 min at room temperature.
Transmission electron microscopy For morphological analysis of cultured cells, samples were grown on 35 mm dishes and fixed using a conventional fixation procedure (see above).
