Polysine microscope slide

Animals were killed by cervical dislocation and hearts were arrested in diastole using a 0.1 mol L⁻¹ cadmium chloride solution (Sigma, Gillingham, UK) before removal from the chest cavity. Whole hearts were weighed, washed briefly in PBS before 24 h fixation in formalin (Sigma) then stored for 72 h in 70% ethanol. The atria were removed and the ventricles dissected into the apex, mid, and base regions and all tissue was processed for paraffin embedding using a Leica TP1020 tissue processor as previously described (Waring et al. 2014 (link)). Five micrometer tissue sections were cut using a Leica RM2235 microtome, mounted onto polysine microscope slides (ThermoFisher, Loughborough, UK), and stored at room temperature until processing for immunohistochemistry.

From 500 to 1,000 HSCs were directly sorted on Polysine Microscope Slides (Thermo Fisher Scientific, P4981-001). Cells were allowed to adhere to the glass slide for 10 minutes, fixed using 4% paraformaldehyde (BD Cytofix, 554655) for 15 minutes, and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich, T9284) for 20 minutes at room temperature. After blocking with 10% donkey serum (Sigma-Aldrich, D9663) for 20 minutes, slides were successively stained with anti-FOXO3 (EMD Millipore, 07-1719, dilution: 1/200) and Alexa Fluor 568–conjugated goat anti-rabbit (Thermo Fisher Scientific, A11011, dilution: 1/500) IgG (H+L) antibodies. After wash, slides were mounted using Gold Antifade Mounting Media with DAPI (Invitrogen, 536939) and analyzed with a LSM 710 confocal microscope system equipped with an inverted microscope (Observer Z1, Zeiss) using ×60 magnification at 1.2 AU pinhole 564 to give an optical section thickness of 0.39 μm images. Image analyses were performed using Imaris (Oxford Instrument, version 9.5) and NIS-elements (Nikon, version 5.20.02) software.

The GFP reporter cells were collected, washed twice, and resuspended in PBS. After exposure to air for 30 min in the dark, 200 μL of cells were added into a 96-well plate, and the GFP fluorescence intensities were measured using a Synergy H4 hybrid microplate reader (BioTek) at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Meanwhile, 40 μL of PBS-resuspended cells were placed on a polysine microscope slide (25 × 75 × 1mm) (Thermo Fisher Scientific, Waltham, MA), covered with a Fisherbrand microscope cover glass slip (15 mm diameter, 0.13 to 0.17 mm thick) (Thermo Fisher Scientific), and visualized under a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems, Buffalo Grove, IL, USA). Excitation was provided at 488 nm, and emission was collected from a range of 500 to 600 nm.

A cryostat (MICROM HM505N) was used to slice 15 μm thick sections. The cryostat was cleaned prior to use. Small plastic molds were used individually for each sample. A small amount of OCT (mounting media for frozen sections) was filled in the mold. A quarter inch of a tubular piece of colon tissue was placed upright and was covered by OCT in the mold, fixed, and frozen. The tissue was allowed to completely freeze at −23°C. The sample was then mounted on slicing stage of the cryotome and sliced into 15 μm thick sections. A paintbrush was used to pull down the desired section of the tissue. The desired slice was then picked up by placing a polysine microscope slide (Thermo Scientific) facedown onto the sectioned tissue. Three to four serial sections were placed on each glass slide. Slides were kept at −23°C and allowed to equilibrate to room temperature prior to hematoxylin and eosinophil (H&E) stain.

After the final maleimide-dye labeling, the bacteria were resuspended in PBS and applied to a custom-made flow cell made of a coverslip attached to a Polysine™ microscope slide (ThermoFisher) by two layers of double-sided sticky tape. After addition of PBS to flush non-adhering cells, the bacteria were fixed by addition of 2% formaldehyde and 0.2% glutaraldehyde in PBS for 5 min. Fluoroshield™ mounting medium containing DAPI (4′,6-diamidino-2-phenylindole) (Sigma-Aldrich) was added. For fluorescent microscopy, a Zeiss Axio Observer microscope at 100 × magnification was used and fluorescence images were analyzed using ImageJ.

Mid-exponential-phase HyPer reporter cells were pelleted, washed twice with phosphate-buffered saline (PBS), resuspended in 100 μl of PBS, and exposed to air in the dark for 30 min. Forty microliters of cells was placed on a Polysine microscope slide (25 by 75 by 1 mm; Thermo Scientific, Waltham, MA), covered with a Fisher-brand microscope glass coverslip (diameter, 15 mm; thickness, 0.13 to 0.17 mm; Thermo Scientific), and then visualized under a confocal laser scanning microscope (Leica model TCS SP8; Leica Microsystems, Buffalo Grove, IL, USA). Excitation was provided at 488 nm, with emission being collected from a wavelength range of 500 to 530 nm (32 (link), 56 (link)). For each sample, at least 5 fluorescent and differential interference contrast (DIC) images were captured. The fluorescence intensities of 25 regions of interest (ROI), with each ROI containing 5 cells, from each sample were measured using Leica Application Suite (LAS) AF software. For images with fluorescence that was too weak, the ROI in the corresponding DIC images was framed, and the fluorescence was measured in the same ROI in the fluorescence image. The average fluorescence intensities of 25 ROIs were calculated and are expressed in arbitrary units (a.u.) per ROI ± standard deviation.

On a Polysine microscope slide (Thermo Scientific, Waltham, MA, USA), a number of brain slices that permitted us to comprehend the entire damage extension were placed. Then, images of the sections were acquired by fluorescent microscopy at a magnification of 4×. The areas of the damage (mm²) on each section were estimated via the “Polygon Selection” tool of ImageJ and following this the total volume of brain damage (mm) was calculated by Cavalieri’s estimator of morphometric volume, which is as follows: VC = d (Σ yi) − (t) Ymax, where d is the distance between the sections contained in a well (d = 240 μm), yi is the area of a single section, t is the section thickness (t = 40 μm) and yMAX is the maximum value of y. The factor (t) yMAX is subtracted from the basic equation as a correction for overprojection.