Tcs sp2 microscope

Frozen sections of large intestine were prepared at 8μm thickness and fixed in 4% paraformaldehyde. Sections were counterstained with 4′6′-diamidino-2-phenylindole dihydrochloride (DAPI) and mounted using Fluoromount-G (Southern Biotechnology). Monoclonal antibodies were as listed in Table 1. Confocal images were obtained using a Leica TCS-SP2 microscope equipped with 405, 488, 594 and 647 nm lasers and image analysis was done in Adobe Photoshop.
Skin was fixed in neutral buffered formalin (Sigma) and tissue sections were stained with H&E for histopathology analysis. Epidermal and scale thickness was quantified as previously described (17 (link)).

Three male apolipoprotein E knockout mouse hearts (ApoE−/−, age 10 months, on a C57BL/6 background, Jackson Laboratory, Bar Harbor, ME) and three, 2 month old female wild type (WT) mouse hearts (C57BL/6 mice, Jackson Laboratory, Bar Harbor, ME) were donated by the Jaffe Lab at Tufts Medical Center. The hearts were stored in 1× PBS during transport, valve isolation and imaging, which occurred within 24 hours from mouse sacrifice. The ApoE−/− mice were fed a high fat diet for 9 weeks before being sacrificed. This model has been previously shown to induce heart valve calcification as measured by von Kossa^{53 (link)} and alkaline phosphatase-positive staining.⁷⁹ TPEF image stacks were acquired at 460 ± 20 nm and 525 ± 25 nm from two distinct regions of the aortic valve of each mouse using the Leica TCS SP2 microscope at an excitation of 800 nm. Each image stack consisted of 5 to 34 optical sections acquired with a step size of 5 μm. The relative MAF contribution for each section was calculated and then averaged from all sections of a given image stack. MAF values for each of the six valves (three ApoE−/− and three WT valves) were calculated by averaging measurements from both Z stacks.