H9c2, WT, 247R, tr247R cells and control Rat1-fibroblasts were plated on glass-bottom plates and fixed for 10 minutes on ice using 1:1 methanol/acetone. Cells were rehydrated, permeabilized with 0.2% Triton X-100 for 5 minutes, blocked with 5% donkey serum, and incubated with anti-vimentin antibody (Dako, Carpinteria, CA, 1:200 dilution) in 1% serum with 0.1% Triton X-100 overnight at 4°C. The next day, cells were washed 3 times 5 minutes in PBS, incubated with secondary antibody (Cy-donkey anti-mouse IgG 1:400 in 1% donkey serum, Jackson Immunoresearch, West Grove, PA) for 45 minutes at room temperature, followed by 3 additional washes with PBS and coverslipping with mounting medium containing DAPI. Images were captured using LSM510Meta scanning confocal microscope (Zeiss, Germany) and a F-Fluar 40x Oil immersion objective (NA = 1.3) under control of Zeiss AIM software, version 4.2, (Zeiss, Germany). The images were created using Zeiss LSM510 software.
Aim software version 4
Manufactured by Zeiss
Sourced in Germany
Zeiss AIM software, version 4.2, is a comprehensive software solution designed to provide advanced image analysis and measurement capabilities. The software's core function is to facilitate the processing, visualization, and quantification of digital images obtained from various microscopy techniques.
Automatically generated - may contain errors
2 protocols using aim software version 4
1
Immunofluorescence Staining of Vimentin
2
Quantitative Analysis of Atherosclerotic Plaque
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At least five to six atherosclerotic-plaques were found on the aortic section of each mouse and then analyzed. The surrounding aortic surface was also included in the analysis. All fluorescence-based and transmitted light images of the sections (Z-Stack images) were acquired with the LSM 510 META Axiovert 200M Zeiss confocal system (Carl Zeiss Meditec AG, Jena, Germany). Images were generated with a 40× Plan-Neofluar 1.3 DIC oil immersion objective (Carl Zeiss Meditec AG) and a multitrack configuration. Hoechst, AlexaFluor488, rhodamine, and Atto655 signals were sequentially monitored with band-pass (BP) 420–480, BP 505–550, and BP 679–743 nm filters after excitation with 405, 488, 543, and 633 nm laser lines, respectively. The Zeiss AIM software version 4.2 (Carl Zeiss Meditec AG) was used for the sequential acquisition of all three channels. All confocal images were acquired with a frame size of 512×512 pixels which was then averaged three times.
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