Meta imaging series 7

Cultures were grown in LB until one hour after the end of the exponential phase. The cells were collected by centrifugation (1 min at 2400×g, room temperature), and washed with 1 ml of phosphate-buffered saline (PBS). Finally, the cells were resuspended in 100 μl of PBS and applied to microscopy slides coated with a film of 1.7% agarose. Images were taken with standard phase contrast and GFP filter, using a Leica DM 6000B microscope equipped with an aniXon + EM camera (Andor Technologies) and driven by Metamorph software (Meta Imaging series 7.7, Molecular Devices). For quantification of the GFP signal, 6 × 6 pixel regions were defined in the desired cell and the average pixel intensity was calculated and corrected by subtracting the average pixel intensity of the background, using Metamorph software (Meta Imaging series 7.7, Molecular Devices).

To estimate the density of BoNT/A-cleaved SNAP-25 labeling, the immunostaining of the L5 spinal sections from rats that received saline, BoNT/A1 or BoNT/A2 was simultaneously carried out in parallel using the same protocols. By means of Meta Morph (Meta Imaging Series 7.0; Molecular Devices, Tokyo, Japan), the optical densities of immunoreactive products were measured on the raw digital images of the ventral horns of the spinal cord. For each animal that received saline (n = 3), BoNT/A1 (10 U; n = 6), or BoNT/A2 (10 U; n = 6), measurements were made in the ventral horns of three spinal sections.

For atherosclerosis experiments, 8-week-old male ApoE KO and ApoE/mitoNEET-Tg mice were fed a high-cholesterol diet (Harlan, TD.88137) for 3 months in a cold-temperature chamber (16°C) with a 12-h:12-h light-dark cycle and free access to water and diet as in our previous study (Chang et al., 2012b (link)). Afterwards, the animals were sacrificed with excess CO₂. After collection of plasma, the mice were perfused with 20 ml normal saline solution through the heart, followed by 20 ml 37% formalin. The mice were fixed with formalin and the whole aortic tree was dissected under a surgical microscope. Next, the aortic trees were stained with Oil Red O solution (0.2% Oil Red O (w/v) in 3.5:1 of methanol:1N NaOH) for 50 min, followed by 70% ethanol for 30 min. Afterwards, the aortic trees were kept in ddH₂O. The attached connective tissues around the aortic trees were cleaned and pinned on a plate containing paraffin wax, and then the aorta was longitudinally opened with a Vannas scissor to expose the atherosclerotic lesions. The pictures of whole aortic trees were obtained using a digital camera, and the atherosclerotic lesion areas were calculated by an Image software (Meta Imaging Series 7.0, Molecular Devices, LLC).