Freshly isolated PMNs (2 × 106 cells/mL) were incubated with control buffer or PAF (10 nM) at 37°C for varying time points during a one hour incubation. After each time point, 40 μL of cell suspension was added to 125μL of HBS containing 0.2% human serum albumin and spun onto glass coverslips using a Cyto-Spin 4 centrifuge (Thermo Scientific, Waltham, MA). PMNs were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton-X-100. PMNs were blocked with PBS/10% goat serum for one hour at room temperature. Antibodies against mTOR, phospho-mTOR ser2448, 4EBP1, phospho-4EBP1 Thr37/46, phospho-4EBP1 Ser65, and phospho-4EBP1 Thr70 were diluted in PBS/10% goat serum and incubated overnight at 4°C. Samples were washed and incubated with a goat anti-rabbit secondary antibody conjugated to the Alexa488 fluorochrome. TOPRO-3 served as a nuclear DNA counterstain. Samples were examined by confocal microscopy using a Fluoview FV1000 confocal microscope and Fluoview software version 4.02 (Olympus, Center Valley, PA). Image quantitation was accomplished using ImageJ image analysis software (NIH, Bethesda, MD).
Fluoview software version 4
Manufactured by Olympus
Fluoview software version 4.02 is a digital imaging solution designed for Olympus confocal microscope systems. It provides a user interface for acquiring, processing, and analyzing images captured by the microscope.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using fluoview software version 4
1
Phosphorylation of mTOR and 4EBP1 in PMNs
2
Phosphorylation of mTOR and 4EBP1 in PMNs
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Freshly isolated PMNs (2 × 106 cells/mL) were incubated with control buffer or PAF (10 nM) at 37°C for varying time points during a one hour incubation. After each time point, 40 μL of cell suspension was added to 125μL of HBS containing 0.2% human serum albumin and spun onto glass coverslips using a Cyto-Spin 4 centrifuge (Thermo Scientific, Waltham, MA). PMNs were fixed with 4% paraformaldehyde and permeabilized with 0.05% Triton-X-100. PMNs were blocked with PBS/10% goat serum for one hour at room temperature. Antibodies against mTOR, phospho-mTOR ser2448, 4EBP1, phospho-4EBP1 Thr37/46, phospho-4EBP1 Ser65, and phospho-4EBP1 Thr70 were diluted in PBS/10% goat serum and incubated overnight at 4°C. Samples were washed and incubated with a goat anti-rabbit secondary antibody conjugated to the Alexa488 fluorochrome. TOPRO-3 served as a nuclear DNA counterstain. Samples were examined by confocal microscopy using a Fluoview FV1000 confocal microscope and Fluoview software version 4.02 (Olympus, Center Valley, PA). Image quantitation was accomplished using ImageJ image analysis software (NIH, Bethesda, MD).
3
Large-scale Confocal Imaging and Analysis
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Samples were imaged using an Olympus FV1000 confocal microscope equipped with a precision motorized stage (Applied Scientific Instrumentation, Inc.). Mosaics were captured using an UPlanSApo 20x oil immersion lens N.A. 0.85 at 1μm intervals in the z-dimension and a pixel display of 800 × 800 along the x-y axes. Image stacks were collected sequentially using the Olympus Fluoview software version 4.2 with 5% overlap between individual tiles. Image registration of individual z-stacks was performed in a semi-automated fashion using the bio-image software Imago 1.5 (Mayachitra Inc.). False color densitometric maps were generated using x,y coordinates from annotated images using the visualization open source package ggplot2 version 3.2.1 as part of RStudio desktop version 1.2.5001 and subsequently rendered using R version 3.6.1. Animal mosaics were quantified under blinding conditions, file names were withheld until after image analysis was completed.
4
Large-scale Confocal Imaging and Analysis
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