Lsm 5 exciter microscope

Neutrophils (1 × 10⁵/300 μL) were seeded in 8-chamber culture slides and incubated with RSV for 180 minutes at 37 °C under 5% CO₂. Afterwards, cells fixed with 4% paraformaldehyde (PFA) were stained with anti-RSV fusion protein (1:1000) followed by anti-mouse PE antibody (1:500), anti-elastase (NE; 1:1000) followed by anti-rabbit Cy3 antibodies (1:500) or anti-myeloperoxidase PE antibody (MPO; 1:1000) and Hoechst 33342 (1:2000). Images were taken in a confocal Zeiss LSM 5 Exciter microscope.

Neutrophils (2 x 10⁵/300 μL) were incubated with F protein (1 μg/mL), LPS (100 ng/mL), PMA (50 nM) or medium alone for 3 h at 37°C under 5% CO₂ in 8-chamber culture slides (BD Falcon). After this period, cells were fixed with 4% paraformaldehyde (PFA) and stained with anti-elastase (1:1000; Abcam), followed by anti-rabbit Cy3 antibodies (1:500; Invitrogen) or anti-myeloperoxidase PE antibody (1:1000; BD Biosciences) and Hoechst 33342 (1:2000; Invitrogen). Confocal images were taken in a Zeiss LSM 5 Exciter microscope.

The slides containing the sections were coded and quantification performed by an experimenter blind to the code. BrdU-positive cells were counted within the SGZ [15] (link) on a Zeiss Axioskop-2 Plus microscope. The number of BrdU-positive cells per section was determined by assessing every sixth section across the rostro-caudal extent of the hippocampus (10 sections/animal). The number of DCX- and GFP-positive cells in the dentate gyrus was quantified (four sections per animal) using a Zeiss Axioscope and Nikon Eclipse 90i fluorescence microscope, respectively. The cell counts are plotted as a percentage of the control (vehicle-treated) for each adrenergic receptor agonist and antagonist. The numbers of cells/section for all treatment conditions are shown in Table S1.
The number of Nestin-GFP/GFAP double-positive cells was quantified by determining the percentage of GFP-positive cells that colocalized with GFAP using confocal microscopy. At least 50 GFP-positive cells from each animal (four sections per animal) were analyzed using z-plane confocal sectioning with 1 µm steps on a Zeiss LSM5 Exciter microscope.

Recombinant mEGFP protein was obtained from ORIGENE (Sku#TP790050). Recombinant mEGFP fusion protein (BRD4aa674–1351; BRD4-IDR–mEGFP) was generated as described previously [35 (link)] and concentrated to an appropriate level using Amicon Ultra centrifugal filters (50K MWCO, Millipore, Burlington, MA, USA). Recombinant protein was diluted to a final concentration of 5 μM in droplet buffer (50 mM Tris–HCl pH 7.5, 10% glycerol, 1 mM DTT, and 10% PEG) containing 10 µM of each aminocyclopropenone, and then immediately loaded on a glass-bottomed dish (MATSUNAMI) and covered with a coverslip. Samples were imaged using a Zeiss LSM5 EXCITER microscope with a mercury lamp and a Plan-Apochromat 100×/1.4 oil objective, as previously described [35 (link)]. Axio Vision software (version 4.8) was used for image acquisition.