Cm1100 cryomicrotome

The frozen intestinal segment was sliced into 12-µm-thick sections at the cross-sectional face using a CM1100 Leica Cryomicrotome (Leica, Wetzler, Germany) at −20 °C. Intestinal sections were thaw-mounted on an indium-tin oxide (ITO)-coated conductive glass slide (Bruker Daltonics) and subsequently dried under nitrogen gas flow.
An ImagePrep automatic matrix sprayer (Bruker Daltonics) was used to spray the matrix uniformly over the ITO glass slide. The following matrix reagents were used: IAA, 1,5-DAN, DHB, and nifedipine at 20 mg/mL in acetonitrile/water (3:1, v/v); 9-AA at 10 mg/mL in acetonitrile/water (3:1, v/v); and nifedipine at 20 mg/mL in acetonitrile/water (3:1, v/v) containing 5 mM phytic acid. The following spraying conditions were used: spray power, 20%; modulation, 20%; spraying time, 1.5 sec; incubation time, 10 sec; drying time, 60 sec; and spraying, 60–70 cycles.

Immunohistochemical analysis of target proteins in the thoracic artery was performed, according to the method used by Idris-Khodja and Schini-Kerth [14] (link). Briefly, segments of thoracic artery were removed and snap-frozen in liquid nitrogen. Frozen aorta was sliced into 14-µm-thick sections at the cross-sectional face using a CM1100 Leica Cryomicrotome (Leica, Wetzler, Germany). The sections were fixed with 4% paraformaldehyde, followed by blocking treatment with 5% BSA in phosphate buffered saline (PBS) containing 0.1% Triton X-100 for 1 h at room temperature. The sections were then incubated with antibodies to AT1R (1∶200 dilution), AT2R (1∶100 dilution) and alpha-1c subunit of Cav1.2 VDCC (1∶100 dilution) overnight at 4°C, following incubation with secondary antibody (Alexa 488-conjugated goat anti-rabbit IgG, 1∶400 dilution) for 2 h at room temperature in the dark. Target proteins on the sections were visualized using a Nikon confocal microscope (Nikon, Tokyo, Japan).