Bx50f4 microscope

The specimens were obtained by direct capture. Additionally, baited pitfall traps were used in the parts of the cave that were considered favourable for the presence of fauna (Fig. 2) (see Luque and Labrada 2016 for details). The specimens were captured using a manual aspirator and then preserved in vials containing 70% ethyl alcohol. Each vial was labelled with the following data: collection site, coordinates, date of capture, name of the organisation, and person involved in the capture. The specimens were mounted in Hoyer medium, and optical observations were made under an Olympus BX51-TF microscope with a multiviewing system and phase contrast, and an Olympus BX50-F4 microscope with differential interference contrast (DIC). For the measurements, a U-DA drawing attachment UIS (Universal Infinity System) and a scale calibrated with a slide by Graticules Ltd (1 mm divided in 100 parts) was used. For electron microscopy, three specimens were fixed with 4% (v/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) for 48 h, then stored for 24 h in a 0.25 M sucrose buffer containing 0.1 M cacodylate, and dehydrated using an ethanol series followed by critical-point drying in CO₂, mounted on aluminium SEM stubs, and coated in Argon atmosphere with 16 nm of gold in an Emitech K550 sputter-coater. SEM observations and photographs were made with a Zeiss DSM 940 A.

The indirect immunoperoxidase staining technique was performed with serial sections (2 μM) of formalin-fixed and paraffin-embedded BM as described [49 (link)-51 (link)]. For MC detection and enumeration, antibodies against KIT (CD117) (polyclonal) and tryptase (mAb G3) were applied overnight. Slides were then washed and incubated with biotinylated anti-rabbit IgG or anti-mouse IgG, washed, and then exposed to streptavidin-biotin-peroxidase-complex. AEC was used as chromogen. The numbers of tryptase+ cells and KIT+ cells (MC) were determined using an Olympus BX50F4 microscope connected to a DP21 camera (Olympus, Hamburg, Germany) and expressed as percent of nucleated BM cells. We also confirmed the presence of MC by Giemsa staining and counted the numbers of MC on Giemsa-stained BM sections in our CML patients.

The SEM images of DBCs and their assemblies were taken using a Hitachi SU-70 Schottky field emission gun SEM at an operation voltage of 10 kV. The confocal microscopy images with a 300 nm voxel depth were acquired using an Olympus FV3000 inverted microscope equipped with a ×60 (NA = 1.42) oil immersion objective (Olympus) and a 532 nm laser. POM images were obtained by an Olympus BX50F4 Microscope with crossed polarizing filters.