Histofix 4

Cell adhesion assay to ECM compound was performed as described previously [43 (link)], using a Reacti-Bind™ amine binding 96-well-plate, coated with fibronectin, collagen I and IV or BSA (all 10 µg/ml) as control over night. On the next day, wells were washed twice with DPBS with 0.05% Tween 20. Unspecific binding sites were blocked with 200 µl blocking solution (DPBS with 0.5% BSA) per well and incubated for one hour in a moistened atmosphere at 5% CO₂ at 37° C in air. Meanwhile cells were detached with trypsin-EDTA and treated for 30 minutes with calcium and/or NPS2143, respectively. Blocking solution was removed and 50 µl of tumor cell suspension (4 × 10⁵ cells/ml) per well were added. After one hour incubation in a moistened atmosphere at 5% CO₂ and 37° C, non-adherent cells were washed out with 2 × 200 µl washing buffer per well. Adherent cells were fixed with 100 µl 4% paraformaldehyde (Histofix 4%, Roth) per well for 15 minutes at room temperature. Adherent and fixed cells were stained using 100 µl crystal violet solution (5 mg/ml in 2% ethanol) for 10 minutes at room temperature. Afterwards the staining solution was washed out and the plate was air-dried. For resolving the colorant wells were incubated with 100 µl 2% SDS (Roth) for 30 minutes on a rocking shaker. The absorbance was measured at 550 nm (reference value at 650 nm).

Cryosections of human healthy and DMD muscles or cultured cells were fixed with Histofix 4% (Carl Roth, Lauterbourg, France) for 10 minutes, permeabilized and saturated with 0.1% Triton X-100/3% bovine serum albumin for 30 minutes, subsequently incubated over night at 4 °C with primary antibody, and then with secondary antibody Alexa Fluor 594 or 488 goat anti-mouse or anti-rabbit IgG (Molecular Probes) for 45 minutes at room temperature. Samples were finally mounted in Mowiol containing DAPI and visualized with an Axiovert miscroscope (Carl Zeiss, Le Pecq, France) under oil immersion. Images were captured and analysed with AxioVision software (Carl Zeiss; Le Pecq, France).

After 1 or 4 weeks, vessels were perfused with India Ink under deep general anesthesia. The abdomen of the animals was opened through a midline laparotomy and the caudal vena cava and aorta were isolated. The caudal vena cava was cannulated, the aorta was severed and the vessels were flushed with heparinized saline solution. Afterwards, a mixture of India Ink (Lefranc-Bourgeois; Colart Deutschland GmbH, Maintal, Germany; 20 mL), saline solution (0.9%; 20 mL), mannite (Carl Roth GmbH & Co. KG; Karlsruhe, Germany, 1.2 g) and gelatin (Carl Roth GmbH & Co. KG; 1.5 g) was injected. The mixture was allowed to cure at −20 °C for 2 h. Subsequently, chambers were transferred to 4% formaldehyde solution (Histofix 4%, Carl Roth GmbH & Co. KG, Karlsruhe, Germany) for 24 h and then processed for histology.