Tcs sp5 laser scanning confocal system

Cryopreserved liver, brain and cerebellum samples from were cut on a cryostat at 14 μm thin sections and placed on glass slides. Sections were brought to RT for 30 min, fixed in 4% paraformaldehyde for 20 min and washed with PBS. For Filipin staining tissues were incubated with 25 μg/mL Filipin (Sigma) over night at 4 °C protected from the light. After washing, slices were mounted with prolong antifade mountant (Dako). For GST-PFO staining, the sections were permeabilized with 0.2% Triton X-100 in blocking buffer (5% goat serum + 1% BSA in PBS) for 2 h in a dark-humid chamber. Then, slices were incubated 3 h with the probe GST-PFO (20 μg/ml) in 1% Goat Serum containing 0.05% Triton X-100 in PBS. After washing × 3 with PBS, samples were incubated O.N. at 4 °C with primary antibody Glutathione-STransferase (GST) (Santa Cruz). Secondary antibodies were diluted 1:200 in 1% Goat Serum containing 0.05% Triton X-100 in PBS and incubated for 90 min at RT with the mix of anti-mouse Alexa fluor-532 (for GST). After washing, slices were incubated 5 min in sudan black 0.1% in 70% EtOH to minimize autofluorescence and mounted with prolong antifade mountant (Dako). Images for all samples were taken with a Leica TCS SP5 laser scanning confocal system with a 633 oil immersion objective APO CS numerical aperture 1.4 equipped with a DMI6000 inverted microscope.

Cryopreserved liver samples from were cut on a cryostat at 14 μm thin sections and placed on glass slides. Sections were brought to RT for 30 min, fixed in 4% paraformaldehyde for 20 min and washed with PBS. The sections were permeabilized with 0.2% Triton X-100 in blocking buffer (5% goat serum + 1% BSA in PBS) for 2 h in a dark-humid chamber. Then, slices were incubated overnight at 4 °C with primary antibodies against Mat1a (PA5115549, ThermoFisher) and ASMase (PA595730, ThermoFisher) in 1% Goat Serum. After samples were incubated O.N, slices were washing × 3 with PBS. Secondary antibodies were diluted 1:200 in 1% Goat Serum and incubated for 45 min at RT with the mix of anti-mouse Alexa fluor-488. After washing, slices were mounted with immersion oil fluorescent free with DAPI (Abcam, ab104139). Images for all samples were taken with a Leica TCS SP5 laser scanning confocal system with a 63x oil immersion objective APO CS numerical aperture 1.4 equipped with a DMI6000 inverted microscope.