Plan apo 20

BMCs were seeded into 48-well plates at a density of 10⁵ cells per well and then cultured in the medium for 24 h to attach. To evaluate the dose-dependent cellular activation of CTSB-PPP, cells were washed and treated overnight, with or without E-64d (50 µM). Cells were washed with PBS, and solutions of CTSB-PPP in DMEM at final concentrations of 6, 12, 24, or 48 µM of Pha equivalents were added for 60 min. In another set of experiments, cells were treated with 6 µM CTSB-PPP for 10 min, 20 min, 60 min, or 24 h. Following treatment, cells were immediately washed twice. The cellular localization of CTSB-PPP was visualized using a confocal microscope (Leica TCS SP5 DMI6000) equipped with a Leica plan apo 20× (numerical aperture 0.7) dry objective. Red fluorescence of the PS and corresponding bright-field images were collected in the fluorescence emission range of 670–700 nm using a 405 nm diode laser for excitation [31 (link),32 (link)]. Pictures were taken using Leica Application Suite Advanced Fluorescence (LASAF) software (Leica Microsystems). Fluorescence intensity per cell was quantified using the ImageJ, software as previously described [33 (link),34 (link)].

The Leica Z6APO macroscope tested is equipped with a DFC290 camera, which is able to take pictures of 3 MP. The objective used was the Leica Planapo 2.0×. The lighting used in this set-up were two Manfrotto ring systems consisting of 24 LEDs each. They are opposed to each other and a diffuser is set within the ring. This entire set-up was set over the specimen. The aperture was set to its maximal opening. The exposure time was positioned according to the colour and reflectivity of the specimen. Setting the start and end positions and well as the other settings for the camera is done with the LAS Core software (http://projects.biodiversity.be/ants).
For the comparison of the different techniques it was necessary to do the stacking with the same software package.