Digital sight ds sm

Monodansylcadaverine (MDC), which is an autofluorescent compound due to the dansyl residue conjugated to cadaverine, has been shown to accumulate in acidic autophagic vacuoles. The concentration of MDC in an autophagic vacuole is the consequence of an ion-trapping mechanism and an interaction with lipids in autophagic vacuoles (autophagic vacuoles are rich in membrane lipids). The use of MDC staining is a rapid and convenient approach by which to assay autophagy, as shown in cultured cells [15 (link)]. Autophagic vacuoles were labeled with MDC by incubating cells on coverslips with 0.05 mM MDC in PBS at 37 °C for 10 min. After incubation, the cells were washed four times with PBS and immediately analyzed by fluorescence microscopy (Nikon Eclipse E 200, Japan) equipped with a blue filter. Images were obtained with a Nikon Digital Sight DS-SM (Nikon, Japan) camera and processed using the program EclipseNet, version 1.20.0 (Nikon, Japan).

Leech tissue samples, dissected from differently treated leech body walls, were embedded in Polyfreeze tissue freezing medium (OCT, Polysciences, Eppelheim, Germany) and immediately frozen in liquid nitrogen. Cryosections (7 µm) were obtained with a cryotome (Leica CM1850, Wetzlar, Germany), collected on gelatinous slides and counterstained with crystal violet and basic fuchsin for morphological analysis. All samples were mounted with Cityfluor (Cityfluor Ltd., London, UK) and examined with a Nikon Eclipse Ni (Nikon, Tokyo, Japan) light microscope. Data were recorded with a Nikon digital sight DS-SM (Nikon).
For ACP assay, cryosections were rehydrated with PBS and incubated with sodium acetate-acetic acid 0.1 M buffer for 5 min, followed by incubation with reaction mixture (sodium acetate-acetic acid 0.1 M buffer, 0.01% naphtol ASBI phosphate, 2% NN-dimethylformamide, 0.06% Fast RedViolet LB and MnCl₂ 0.5 nM) for 90 min at 37 °C. After several washings in PBS, slides were mounted with PBS/glycerol and observed with a Nikon Eclipse Ni (Nikon) as above.