Uplanapo

Wild type (WT) and various mutants were observed with an inverted microscope (Willowert Wetzlar 21-D6330, X4 objective), and photographed (X10 objective) with a BX50 Olympus microscope equipped with a differential interference contrast (DIC) device and a digital camera (Olympus F-view II). When necessary, a small piece of agar supporting several colonies was covered with a glass cover slip, cut and placed on a microscope slide to observe the presence of cysts or rods with the 100 X oil immersion objective (UPlanApo, Olympus)^{4 (link)}.

Spermatocyte squashes were obtained as described previously (Page et al. 1998 (link), Viera et al. 2002 (link)). After fixation, squashed spermatocytes were rinsed three times in PBS for 5 min, and incubated with mouse anti-SYCP3 (1:100), ACA/CREST serum (1:30) and guinea pig anti-SKAP (1:100) antibodies diluted in PBS at 4 °C overnight. Nuclei spreads were prepared according to the drying-down technique as described in Peters et al. (1997) (link) and immunostained as described in Grey et al. (2009) (link). For studies in HeLa cells, cells were grown on coverslips and transfected with GFP-SKAP overnight. For immunofluorescence, HeLa cells were washed with PBS, fixed in methanol for 10 min and permeabilized in PBS/0.05% Triton X-100/5% fetal veal serum for 1 h. Cells were incubated with primary antibodies (rabbit anti-GFP, 1:1000 and mouse anti-tubulin clone DM1a, 1:500) in blocking solution at room temperature (RT) for 1 h. After washes in PBS containing 0.1% Tween-20 (PBS-T), cells were incubated with secondary antibodies for 1 h at RT and then stained with DAPI (1:2000). Images were recorded with a Zeiss Axioimager Z1 microscope equipped with a Coolsnap HQ2 camera at 1×1 binning with a 100×, 1.3 NA Olympus U-PlanApo objective. Images were imported into Image J for further processing.

Human Embryonic Kidney 293 cells stably expressing B₂R-GFP at the level of their plasma membrane exhibit BK-induced endosomal internalization of the fluorescent receptor, maximal 30 min after stimulation but with gradual recycling to the cell surface in 1–3 h (Bachvarov et al., 2001 (link); Charest-Morin et al., 2013b (link)). A microscopic assay of extended BK analogs was based on this system and on the fact that the culture medium containing 10% heat-inactivated fetal bovine serum contains the active soluble form of ACE that remains the main BK inactivation pathway in the system (Bachvarov et al., 2001 (link)). Arg-CP(s) sensitive to Plummer’s inhibitor are also found in human plasma and inflammatory synovial fluid that contains serum proteins (Chercuitte et al., 1987 (link)), suggesting that serum-containing culture medium may contain this enzymatic activity. HEK 293 cells stably expressing B₂R-GFP were observed in epifluorescence microscopy at a 1000× magnification and photographed using an Olympus BX51 microscope coupled to a CoolSnap HQ digital camera (filters for GFP and fluorescein: excitation 460–500 nm, emission 510–560 nm, objective lens 100× oil UPlanApo, Olympus). The experiments were based on BK or BK analog stimulation of different durations to monitor either the endocytosis of B₂R-GFP (30 min) or their recycling (3 h), based on previous time course findings.

The frozen cardiac tissue of ACT patients, as well as healthy controls, was embedded, sliced, and stained with Masson’s trichrome staining in cooperation with the Department of Pathology of the University Medical Center Göttingen. The area of fibrosis was quantified in cooperation with the Technology Platform Clinical Optical Microscopy. The slides were first scanned for virtual microscopy using a 20 × objective (UPlanApo) with the dotSlide SL slide scanner (Olympus) and a peltier-cooled XC10 camera. Fibrotic areas were then calculated by a scientist blinded to analyzed myocardium, using the CellSens Dimensions software (Olympus).

The morphologies of the IM bulk powder and the IM microparticles without PVA were characterized using an optical microscope (OLYMPUS BX51, Olympus Corporation, Tokyo, Japan) equipped with a microscope digital camera (Visualix V500FL, Visualix K.K., Hyogo, Japan). A 40× magnification objective lens (UplanApo, Olympus Corporation) was used. The morphologies of the IM microparticles (IM-NK(50), IM-KP(50), IM-NK(Dry), IM-KP(Dry)) were characterized using a scanning electron microscope (SEM). The JSM-6060LA scanning electron microscope (JEOL Ltd., Tokyo, Japan) was used to obtain SEM images.

Other recipient HEK 293a cells were grown and transiently transfected as described above with a vector coding for the human B₁R C-terminally tagged with FLAG (hB₁R-FLAG) [14 (link)], a pharmacologically intact B₁R construction, or with human B₂R or ACE fused at their C-terminus to the mCherry FP. Stimulations for microscopic or cytofluorometric experiments were based on the lysates for the GFP/mCherry-based ligand constructs. Receptor- or ACE- expressing HEK 293a cells were generally treated for 30 min with stimulants (incubation carried out at 37°C in humidified atmosphere containing 5% CO₂), rinsed 3 times with phosphate buffered saline, observed in microscopy for epifluorescence and photographed using an Olympus BX51 microscope coupled to a CoolSnap HQ digital camera (filters for GFP: excitation 460–500 nm, emission 510–560 nm; for mCherry FP: excitation 525–555 nm, emission 600–660). The objective lens was the 100× oil UPlanApo (Olympus).