Confocal software v 2

DAB immunohistochemistry was examined and recorded using a Nikon Eclipse E600 microscope equipped with a DMX2000 digital camera (Nikon, Tokyo, Japan). Maps of antigen distribution were constructed using a camera lucida tube attached to a Zeiss Axioskop microscope (Zeiss, Munich, Germany) at 20× magnification. These were scanned and reduced to the final size for reproduction. Confocal immunofluorescence was analyzed with a laser confocal scan unit TCS-SP8 equipped with argon and helio-neon laser beams attached to a DMIRB inverted microscope (Leica Microsystems). For Cy3 fluorophore, excitation was 433 nm for 560–618 nm emission. For Alexa 488, excitation was 488 nm for 510–570 nm emission. Serial 0.3 μm sections were imaged to assess colocalization of relaxin-3-immunoreactive terminals with synaptophysin, using Leica Confocal Software (V 2.61).
In n = 3 cases, the co-localization of synaptophysin and relaxin-3 was analyzed in different amygdala divisions. Captured images using the 40× objective were taken through a total depth of 10 μm and then reconstructed with the Leica application suite (LAS-X) software. Firstly, we studied the percentage of relaxin-3 fibers that does not co-localize with synaptophysin. Second, we studied the density of synaptophysin boutons along the length of relaxin-3 fibers.

Confocal imaging was undertaken, at room temperature, in pairs (or multiples thereof) so that one Crohn’s disease section was always accompanied by a control section, and identical laser intensities were used. Detector gains for Crohn’s disease sections were set equal to, or higher than, that of the matched control ensuring that even faint PD-L1 staining above background could be detected for the former. Sections were imaged with a Leica DMIRE2 microscope (Leica Microsystems, Germany) at 488, 568 or 633 nm, fitted with diode Ar/ArKr and HeNe lasers, using either a x20, 0.7 NA multi-immersion lens or a x63, 1.2 NA water objective lens. Data were recorded using the Leica Confocal Software (v2.61) and images processed using ImageJ30 (link). Data were collected as 8-bit greyscale images and subsequently assigned appropriate colours. Nuclei are shown grey to enable better visualisation of non-nuclear stains. Identical imaging and data collection routines were applied to Crohn’s disease and control sections. Three-dimensional representations were deconvoluted and reconstructed from z-stacks using Huygens Professional deconvolution software (SVI, The Netherlands).

The procedure was described in our recent papers (Jukkola et al., 2012 (link), 2013 (link)). High-magnification confocal images were captured with a Leica TCS SL confocal imaging system (Leica Microsystems, Mannheim, Germany), using a 100× HCX Plan Apo CS oil immersion objective (numerical aperture = 1.40). Each image was averaged over four scans in linescan mode. Images were saved as 8-bit TIFF files and adjusted for brightness and contrast using Adobe Photoshop 7.0. For 3D reconstruction, z-stacks of images were collected with a 0.5-μm slice interval. Collapsed images were generated using a maximum intensity projection of the z-stack. For each 3D image, the z-stack was visualized in three-dimensional cross sections, and the cross-bars were centered on pertinent features of the image. The three one-dimensional images were then exported together as an 8-bit TIFF file. Supplementary movies were created using the Maximum Projection with Animation tool, and were adjusted for brightness and contrast within the Leica Confocal Software (v2.61). A maximum intensity projection of the z-stack was rotated 90 degrees around the Y-axis in a series of 18 steps and exported as an AVI file.

Concentrations of ERY, CIP, and TASA used in this assay were 500 μg/mL, 500 μg/L, and 20 mg/mL, respectively. For CLSM analysis of biofilm, above treated bacteria cultured on glass cover slides were incubated with 5 μL DNA fluorescent staining solution (the fluorescent staining solution was mixed by SYTO9 and propidium iodide prior to be applied). The plate was incubated at 37°C for 15 min in dark. After washing for 3 times with PBS, the aseptic cover glass was placed on a glass slide [20 (link)]. The stained biofilm was then examined with CLSM with parameters of green exciting light at 488 nm, red exciting light at 543 nm, 40x objective, and 10x eyepiece using a Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 (Leica, Germany). Each group was repeated three times and 3 different fields were randomly selected for each sample. Then the selected fields were scanned layer by layer from 4 to 20 layers based on the thickness of biofilm. Viable bacteria were displayed in green fluorescent, dead bacteria were imaged in red fluorescent, and an area with both viable and dead bacteria was superimposed as aurantiacus fluorescence in images of CLSM.

M. ovipneumoniae was cultivated until the late stationary growth phase, and the sample was then pelleted by centrifugation for 30 min at 12000 × g. After washing three times for 10 min with phosphate-buffered saline (PBS, pH = 7.4), bacterial cells were fixed with 4% paraformaldehyde for 15 min followed by a permeabilization with 0.3% TritonX-100 for 10 min at room temperature. Nonspecific antibody binding was subsequently blocked using 3% normal BSA in PBS for 2 h at room temperature, after which rabbit anti-M. ovipneumoniae CPS IgG (self-made antibodies) (1 : 100) and rabbit nonspecific IgG (Beyotime, Shanghai, China) were applied and incubated in a humidified chamber at 4°C overnight. The binding of primary antibody was then detected by an addition of Rhodamine- (TRITC-) labeled goat-anti-rabbit IgG secondary antibody (Proteintech Group, Chicago, USA) (1 : 100). After extensively washing for 3 × 5 min, bacterial cells were mounted on slides and photographed using LEICA TCS SP2 A0BS Confocal system. Images were processed by Leica Confocal Software v.2.6.1 (Leica, Mannheim, Germany).

Immunofluorescent staining was applied to determine the expression of AECII cell marker surfactant protein C (SPC) and AECI cell marker aquaporin-5 (AQP-5). The membranes of 2-week ALI cultures were fixed in filtered 4% paraformaldehyde at room temperature for 15 min prior to be washed for 3×3 min with PBS. The cells were then permeabilized with 0.2% Triton X-100 for 20 min at room temperature, followed by blocking with 5% normal donkey serum in PBS at room temperature for 60 min, after which they were incubated with primary antibodies against SPC (1:1000, Merck Millipore, USA) or AQP-5 (1:500, Abcam, USA) in PBS at 4°C overnight. Following extensive washing for 3×10 min with PBS to remove primary antibodies, the membranes were incubated with Alexa Fluor 488-labelled donkey-anti-rabbit IgG secondary antibody (1:500, Jackson ImmunoResearch Laboratories, USA) at room temperature for 60 min. The stained membranes were then mounted on slides with Vectashield Mounting Medium containing DAPI (Vector Laboratories, USA), and covered with a coverslip after washing in PBS for 3×5 min. Images were acquired by Leica TCS SP2 A0BS Confocal System and processed on Leica Confocal Software v.2.6.1 (Leica, Germany).