Evos fl auto imaging system software

SMA tissue was collected for proteomic and histological analysis. Mice that were designated for proteomic quantification were euthanized by CO₂ asphyxiation, and the SMAs were collected. The adventitia and periadventitial fat from the SMAs were removed in cold PBS; then the SMA was immediately snap-frozen and stored at –80°C until analysis. For histological analysis, SMAs were collected after micro-CT images were acquired. Residual Microfil contrast reagent was removed from the SMA. The tissue was then fixed again in 4% paraformaldehyde overnight, then placed in 70% ethanol solution in preparation for decalcification, sectioning, and staining. Tissue cross sections of 5 μm thickness were sliced and stained by H&E or EVG. Morphometric measurements were performed using EVOS FL Auto Imaging System software (Invitrogen, Thermo Fisher Scientific) and ImageJ (NIH). All measurements were performed while blinded to the sample identification.

Polyps were incubated for 30 min in 1 μg/mL propidium iodide in HM, washed twice in HM, then mounted on glass slides as described for live imaging of neurons. Slides were imaged on an Invitrogen EVOS FL Auto microscope in the red fluorescence channel using the Invitrogen EVOS FL Auto Imaging System software. Labeled cells were counted in the body column only and reported as number of labeled cells per animal. As a positive control, polyps were incubated in 0.04% colchicine (Acros Organics) in HM to induce cell death [98 (link)]. Animals were incubated in colchicine for a full 24 h rather than 8 h incubation followed by 16 h recovery as described.

The whole neck and head were dissected from LRP1^+/+ and smLRP1^–/– mice subjected to left carotid artery ligation with and without losartan. Samples were then skinned and fixed in 10% buffered formalin phosphate (fixative solution; Thermo Fisher Scientific SF100-20) for 3 days, with fixative solution exchanged for fresh fixative solution once per day. After 3 days of fixation, samples were placed in 70% ethanol solution and transferred to the Center for Vascular and Inflammatory Diseases Histology Core at the University of Maryland School of Maryland or shipped to Histoserv, Inc. for decalcification, sectioning, and staining. Whole-neck serial cross sections of 5 μm thickness were sliced starting from the carotid bifurcation to the area inferior to the lesion apex. The apex of the lesion area was identified by analyzing serial sections at 100 μm intervals by H&E, EVG, and Masson’s trichrome staining. Morphometric measurements were performed using EVOS FL Auto Imaging System software (Invitrogen, Thermo Fisher Scientific). All measurements were performed while blinded to the sample identification.