Lsm 5 pascal system

Brain sections from 10-month-old 5XFAD/apoE^m/4 or 85-day-old APPPS1-21/apoE^m/4 mice were co-stained for fibrillar amyloid, mouse apoE, and apoE4 using X-34, anti-mouse apoE monoclonal antibody HJ6.3-Alexa Fluor 568 (generated in-house) and anti-human apoE monoclonal antibody HJ15.7-Alexa Fluor 488 (generated in-house), respectively. For quantification of apoE/CAA or apoE/plaque co-staining in 10-month-old 5XFAD/apoE^m/4 mice, brain sections were imaged using a Zeiss LSM 5 PASCAL system coupled to a Zeiss Axiovert 200 M confocal microscope. Three sections from each mouse were used and five fields containing CAA and five fields containing plaques on each section were imaged. Images were thresholded for corresponding colors and the degree of co-localization was analyzed with ImageJ. Percent area of CAA or plaque covered by mouse apoE or apoE4 was calculated and the average from the 3 sections/mouse was used to represent each mouse. For quantification of apoE/plaques in 85-day-old APPPS1-21/apoE^m/4 mice, which were at the initiation of plaque deposition, all the plaques in the cortical area were counted under Nikon Eclipse 80i fluorescent microscope and the apoE co-staining status for each individual X-34 plaque was recorded. The average of 3 sections from each mouse was used to represent each mouse.

Image acquisition was performed using an LSM710 system (×40, NA 1.2 water immersion objective or ×63, NA 1.4 oil immersion objective; Zeiss), an LSM5 PASCAL system (×63 NA 1.2 and ×20 NA 0.60; Zeiss), an A1Rsi system (×63 NA 1.3 water immersion objective; Nikon) or a TSC SP8 system (×40, NA 1.10 water immersion objective; Leica). Bright field images were acquired by detecting transmitted light (488 or 552 nm). For live-imaging, Xenopus gastrula embryos were embedded with 1.5% LMP agarose (#16520-050; Invitrogen) gel in 1/10× Steinberg’s solution on 35 mm glass-based dishes (Iwaki) or mounted in a house-made imaging chamber. For confocal imaging of immunostaining, stained embryos were mounted in shallow wells on 2% agarose plate, flattened with a coverslip. Images were cropped and/or processed with Photoshop CS4 (Adobe) or ImageJ (NIH). Fluorescent intensity was measured using Image J (NIH) or Zen (Zeiss).

Confocal images were generated on an LSM 5 Pascal system (Zeiss, Thornwood, NY, USA) equipped with 40X 1.3 NA and 63X 1.4 NA objectives.

HFFs were seeded in 4-well chamber slides to a confluence of approximately 70%. They were mock-infected or infected with HSV-1 at an MOI of 3. After 1 h, the inoculum was replaced by medium with 0.1% DMSO or 10 µM NFV, and the cultures were returned to the incubator. Sixteen hours after infection, the cells were fixed in 4% paraformaldehyde in phosphate-buffered saline. Cells were then permeabilized with 0.2% Tween-20. Endogenous peroxidase activity was inhibited with 3% H₂O₂. The cells were then incubated with 10% nonfat dry milk containing 1% normal goat serum (blotto/NGS; Jackson) to block nonspecific binding. Primary antibody binding was revealed with anti-mouse IgG-HRP (Jackson) followed by a 10-minute TSA amplification with TSA-594 (Life Technologies). The nuclei were stained with TO-PRO 3. Slides were mounted after addition of SlowFade (Life Technologies) and analyzed by confocal microscopy. Confocal images were generated on an LSM 5 Pascal system (Zeiss).