Axiovert 40 cfl inverted microscope

This assay was performed according to the protocol of Baker and Robinson^{54 (link)}. In summary, thoracic aortae were dissected from mixed background mice, cut into rings approximately 0.5 mm in width and incubated in serum-free Opti-MEM (Gibco, cat. no. 51985026) at 37 °C overnight. Each ring was then embedded in separate wells of a 96-well plate containing 1.2 mg/ml of collagen I (Millipore, cat. no. 08115), which was polymerised by leaving the plate at 37 °C for 30 mins. Rings were fed with fresh Opti-MEM supplemented with 2.5% FBS and VEGF (30 ng/ml) at 37 °C every 3 days with different doses of SFN. After 6 days, rings were fixed with 4% (v/v) formalin and the microvessel counted by phase-contrast microscopy. To further staining, specific epitopes can be fluorescently labelled. After the fixing, the rings were permeabilised with 0.25% (v/v) Triton X-100 in PBS, and blocked with 2% (v/v) BSA in PBLEC (PBS with 1 mM CaCl₂, 1 mM MgCl₂, 0.1 mM MnCl₂, 1% Tween-20) for 30 mins at 37 °C. FITC-conjugated BS-1 lectin (Sigma, cat. no. L9381/L5264) was used to stain endothelial cells and anti-actin α-smooth muscle Cy3 (α-SMA; Sigma, cat. no. C6198) was used to stain the supporting cells. 1 µg/ml DAPI was used to stain the nuclei. Images were taken by Axiovert 40 CFL inverted microscope (Carl Zeiss).

HUVECs (Invitrogen, Waltham, MA USA) were plated at a high concentration (124,000 cells/well) on 24-well culture plates (Corning Life Sciences, Glendale, AZ, USA) that had been coated with Matrigel^® (300 μL/well, 9.5 mg/mL, BD Pharmingen, San Diego, CA, USA) and maintained at 37 °C for 16 h in Human Large Vessel Endothelial Cell Basal Medium (formerly M200, Gibco/ThermoFisher Scientific, Waltham, MA, MA), supplemented with a high growth factor supplement kit (ATCC). The plates were checked to confirm a two dimensional capillary-like network had formed, and tube disruption was induced by the addition of varying concentrations of OXi6196 or colchicine (10 nM, 100 nM, and 1000 nM) for 2 h. Medium was removed and the cells were washed twice with M200. The plates were photographed (nine fields per well at 5× magnification) using a Canon Powershot A640 digital camera mounted onto an Axiovert 40 CFL inverted microscope (Zeiss, Thornwood, NY, USA) [31 (link)].

Cells were washed 2 × 5 min in phosphate buffered saline (PBS) and then fixed with 4% paraformaldehyde/PBS for 15 min. Cells were then washed in PBS (3 × 5 min), permeated with 0.1% TritonX-100/PBS, washed in PBS (3 × 5 min) and then incubated in 3% bovine serum albumin (BSA)/PBS for 2 h. Cells were incubated with primary antibody overnight at 4°C. MF20 (1:50; Developmental Studies Hybridoma Bank, University of Iowa, Department of Biology, Iowa City, IA, USA) in 3% BSA/PBS was used to stain myosin heavy chain (MHC). Cells were then washed with PBS (3 × 5 min) and incubated in goat-anti-mouse IgG2b Alexa555 secondary antibody (1:500, Life Technologies) and DAPI (1:1,000) for 2 h in 3% BSA/PBS. Cells were washed in PBS (3 × 5 min) and then imaged on a Zeiss Axiovert 40 CFL inverted microscope using a 10X objective. Four images were taken in each well from pre-defined locations within each quadrant. Myotube diameter was measured using AxioVision software (AxioVision AC Rel. 4.8.2, Carl Zeiss Imaging Solutions, Wrek, Göttingen; Germany). A total of ~50–80 myotubes were measured per well and the average diameter of each well was used for statistical analysis as described previously (6 (link), 9 (link), 10 (link)).

After METH administration and Ag sensitization, each mouse was euthanized and lung/spleen tissues were excised and fixed in 4% paraformaldehyde (Sigma) for 24 h. Tissues were processed, embedded in paraffin, and 4 μm sagittal sections were fixed to glass slides. The tissues were then subjected to hematoxylin and eosin (H&E) staining, CD4- (clone W3/25)^{44 (link)}, and CD8- (clone 32-M4)^{45 (link)} specific antibody (Ab; conjugated to horseradish peroxidase; dilution: 1:1000; Santa Cruz Biotechnology; SCB) immunostaining to assess tissue morphology, helper T cell, and cytotoxic T cell infiltration (brown), respectively. The slides were visualized using an Axiovert 40 CFL inverted microscope (Carl Zeiss; CZ), and images were captured with an AxioCam MRc digital camera using the Zen 2011 digital imaging software. The hyperplasia of type II pneumocytes was recorded microscopically in single cuboidal cells showing large nuclei, prominent nucleoli, and scant to abundant cytoplasm. The infiltration of CD4⁺ and CD8⁺ cells to pulmonary and splenic tissue was quantified by cell counts using the recorded 40 × images. Each image was blindly analyzed by three independent investigators. Ten microscopic fields per image were counted and the average was calculated per image (n = 10 images; 10 fields per image; 2 images per mouse; 5 mice per condition).

Confluent monolayers of TRAF2^+/+ and TRAF2^−/− MEFs in a 6-well plate were scored using a 200-μl pipette tip to generate wounds devoid of adherent cells. Monolayers were infected at an MOI of 5 for 1 h on ice with VACV A5L-EGFP and then incubated at 37°C. At the times indicated below, the monolayers were examined using a Zeiss Axiovert 40 CFL inverted microscope and the number of cells which had migrated into the wound in each well was counted. Representative images were captured using a Canon Powershot A640 camera.