Eclipse e400 fluorescence microscope

Forty-eight hours after transfection with pre-miRNA precursors for hsa-miR-103a, hsa-miR-585-5p, or Scr control, HAECs were untreated or treated with 100 μM H₂O₂ for 30 min in serum-free EMB-2. This concentration of H₂O₂ induces various base lesions and DNA breaks. Single-cell alkaline comet assays were performed essentially as described [33 (link), 34 (link)]. Comets were imaging on an Eclipse E-400 fluorescence microscope (Nikon, Japan) attached to a Pulnix video camera (Kinetic Imaging, LTD, Liverpool, UK) and were quantified using Komet 5.5 software (Kinetic Imaging LTD). Olive tail moment was used as a measure of DNA damage level [35 (link), 36 (link)].

Cells were passaged on glass slides coated with poly-L-lysine and left to adhere for 24 h. Fixation was done in 3.7% formaldehyde/PBS at RT for 20 min, followed by extensive washing with PBS. Nonpermeabilized samples were directly incubated with tested antibody variants, whereas cells intended to be permeabilized were treated with 0.1% Triton X-100/PBS for 20 min and washed with PBS beforehand. Antibody variants were diluted to 14 µg/mL in PBS and incubated with slides at 4 °C overnight. Binding was detected using either anti-6× His tag antibody (2 µg/mL in PBS for 1 h; Sigma) or anti-Strep tag Ab (2 µg/mL in PBS 2 h; Immo, IBA) combined with a goat anti-mouse IgG Alexa Fluor 488 conjugate (4 µg/mL in PBS/0.05% Tween-20 1 h; Thermo Fisher Scientific). All incubation steps were followed by extensive washes with PBS/0.05% Tween-20. Finally, cells were counterstained with 6-diamidino-2-phenylindole (DAPI; 1 µg/mL) for 5 min and mounted in VectaShield (Vector Laboratories, Burlingame, CA, USA). The fluorescence signal was visualized by an Eclipse E400 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a 20× and 40× magnifying dry objective. Images were taken by a ProgRes MF CCD camera (Jenoptik Optical Systems GmbH, Jena, Germany) and processed in Adobe Photoshop software.

For yeast cell cycle analysis, 1 × 10⁷ cells were incubated with PMA, Roy-Bz, or vehicle, for 42 h. Cells were fixed and stained with 10 μM Sytox Green (Invitrogen, Alfagene, Carcavelos, Portugal) followed by flow cytometry analysis^{16 (link)}. Propidium iodide (PI; Sigma-Aldrich) and TUNEL staining, using In Situ Cell Death Detection Kit Fluorescein (Roche Diagnostics, Sigma-Aldrich), were used to monitor necrosis and apoptosis, respectively^{16 (link)}. Approximately 500 cells were counted in five random microscope fields using an Eclipse E400 fluorescence microscope (Nikon).

Sections of paraffin-embedded small intestine biopsies were obtained using a Leica SM 2000R microtome; subsequently they were rehydrated and Citra solution (Biogenex, cat. HK086-9K) was used for antigen retrieval. Sections were stained with anti-IFABP or anti-LFABP rabbit polyclonal antibodies (dilutions used: LFABP 1/200–1/400, IFABP 1/20–1/50). Afterwards, they were incubated with anti-rabbit IgG conjugated with Alexa 488 (Life Technologies, cat. A-21206). Nuclei were stained with propidium iodide or DAPI. Confocal fluorescence microscopy analysis was performed in a SP5 Leica confocal microscope. Immunofluorescence analysis was performed in a Nikon Eclipse E400 fluorescence microscope.

Human embryonic kidney (HEK) 293 cells were cultured in high-glucose Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum, as described (17 (link)). WT (Leu723) and mutant (Gln723) CaSR-pEGFP-N1 constructs were generated, as reported (16 (link)), and transiently transfected into HEK293 cells using Lipofectamine Plus (Invitrogen), as described (16 (link)). Successful transfection of WT and mutant CaSR proteins was confirmed by visualizing green fluorescent protein (GFP) fluorescence using an Eclipse E400 fluorescence microscope with an epifluorescence filter, and images were captured using a DXM1200C digital camera and NIS Elements software (Nikon, Tokyo, Japan), as described (17 (link)).