Ix83 microscope

Fluorescence microscopy was performed using an Olympus IX83 microscope equipped with a ×100/NA 1.40 (Olympus) objective and an Orca-R2 cooled CCD camera (Hamamatsu), using Metamorph software (Universal Imaging). Simultaneous imaging of red and green fluorescence was performed using an Olympus IX81 microscope, described above, and an image splitter (Dual-View; Optical Insights) that divided the red and green components of the images with a 565-nm dichroic mirror and passed the red component through a 630/50 nm filter and the green component through a 530/30 nm filter. Dual color time lapse imaging of red and green fluorescence was performed using an Olympus IX83 microscope equipped with a high-speed filter changer (Lambda 10-3; Shutter Instruments) that can change filter sets within 40 ms. Images for analysis of colocalization were acquired using simultaneous imaging (64.5 nm pixel size), described above. Intensity profiles of GFP-fused protein and mCherry/tdTomato-fused protein were generated across the center of fluorescence signals used for the assessment (representative intensity profiles are shown in Figs. 4g, 5a, Supplementary Figs. 1a and 3b). Colocalization was defined as occurring when the distance between the two peaks of GFP and mCherry/tdTomato intensities was <129 nm (2 pixels).

The cultured
cells were gradually fixed with PFA (10 min 1% and 10 min 2%) and
permeabilized for 5 min in PBS containing 0.2% Triton-X100. For focal
adhesion (FA) staining, anti-vinculin FITC conjugated mouse monoclonal
antibody (dilution 1:50; F7053 Sigma Aldrich) was used and incubated
for 1 h at room temperature followed by imaging with an Olympus IX83
microscope (λ_ext at 470 nm, λ_em at
525). Data represents the mean number of FAs per one cell of ∼60
randomly selected adherent cells, calculated using the ImageJ software.
According to the attached protocol, cytoskeletons were stained using
ActinGreen 488 Ready Probes (Life Technologies R37110). Images were
taken using an Olympus IX83 microscope (λ_ext at 470
nm, λ_em at 525).

Neonatal lungs sections were collected and fixed with 4% PFA and stored. Samples were then heated to deparaffinize and rehydrated with xylene and ethanol.
For DAB immunohistochemistry slides were quenched with BLOXALL (Vector labs) and blocked. They were then incubated with IL-1α primary antibodies (R&D systems, AF-400-NA), followed by a biotinylated secondary antibody, VECTASTATIN. R.T.U. ABC Reagent, and finally stained with DAB peroxidase (Vector Labs). They were counterstained with methyl green. IL-1α staining was visualized using the Olympus IX83 microscope and Olympus DP80 camera at Å~40 magnification using Olympus CellSens software.
For immunofluorescence, antigen retrieval was performed with citric acid, tissues were permeabilized with 0.5% triton, quenched with glycine, and blocked. They were incubated with IL-1α (R&D systems, AF-400-NA) and pro-surfactant protein C (Millipore, AB3786) primary antibodies overnight followed by incubation with secondary antibodies (Alexa fluor anti-rabbit donkey 647 and anti-goat donkey 594) for 1 h. Staining was visualized using the Olympus IX83 microscope and Olympus DP80 camera using Olympus CellSens software.