Dm5500b epifluorescence microscope

GlyT2+ boutons located on LSO and SPN neuron somata of P7, P12 and P14 mice were counted on six sections/side (N = 12 sections/mouse) on photographs taken at 60 μm increments on a Leica DM5500B epifluorescence microscope. For adult mice, bilateral LSO and SPN images across 10 sections/side (N = 20 sections/mouse) at 50 μm increments were used for counting. Images were processed using ImageJ 1.47V (NIH), and the number of boutons on all neuronal soma with an identifiable nucleus were counted and reported as mean puncta per neuron ± SEM. For analyzing localization of glycinergic innervation in the LSO or SPN, boutons were measured by distance from the cell body using Volocity software. The origin of the dendrite (0 μm distance from the soma) was defined as the region where the soma narrowed to form a distinct process. Neurons with an identifiable nucleus and visible dendrites at least 10 μm in length were used for analysis (N = 12 dendrites/genotype/brain region). Glycine receptor α1 isoform (GlyRα1) clusters were quantified by counting the numbers of immunopositive puncta located on LSO or SPN neuronal somata (N = 4 sections/mouse at 50 μm increments).

Age-synchronized adults were placed on NGM plates seeded with the E. coli OP50-1 and containing 100 μM MitoTracker Deep Red FM (Thermofisher) or 100 μM TMRE (tetramethylrhodamine, ethyl ester) (Biotum). The animals were kept on the plates for 24 h in the dark and then recovered on regular plates for 2 h. Animals were then fixed with 4% paraformaldehyde and imaged using a LEICA DM5500 B epifluorescence microscope. MitoTracker was imaged using a × 40 or a × 60 numerical aperture objective with a 633-nm line for excitation. TMRE was imaged using a × 10 numerical aperture objective with a 549-nm line for excitation. TMRE staining was quantified using CellProfiler cell image analysis software. The numbers of individuals examined (n) in each condition tested are noted in the figure legend of Additional file 1: S1B. Individual data values are included in Additional file 2.

Overnight cultures in TSB were diluted to an OD₆₀₀ of 10. Untreated samples were taken from the overnight culture. Convertase-covered cell samples were obtained as described above. Cells were collected by centrifugation at 14.000 rpm for 2 min and washed with PBS. The washed cells were incubated with the 6D4–800CW (3000 ng/mL in PBS) for 30 min. After the incubation, the cells were collected by centrifugation at 14,000 rpm for 2 min and washed with PBS. Next, cells were spotted on a glass slide for microscopy, and a coverslip was mounted and sealed. Fluorescence microscopy was performed using a Leica DM5500B epifluorescence microscope equipped with an 800 nm filter block. Images were captured with a Leica DFC365FX camera using a 63x objective (Leica Microsystems BV, The Netherlands).

Aliquots of 1 ml of bacterial cultures (OD₆₀₀ of 1) in TSB were collected, washed twice with PBS, and resuspended in 1 ml of PBS. Next, ~10¹⁰ cells thus obtained were incubated with 0.65 mg/ml IRDye 800CW-labeled 1D9 or 1D9 F(ab’)₂ for 1 h at room temperature. After washing 3 times with PBS, cells were spotted on poly-L-lysine-coated glass slides (Sigma Aldrich) and inspected using a Leica DM5500B epifluorescence microscope equipped with an 800 nm filter block. Images were captured with a Leica DFC365FX camera using a 63× objective (Leica Microsystems BV, The Netherlands).

The method of immunofluorescence technique for fibrinogen expression was performed according to the modification of Abdel-Bakky 2022 [47 (link)]. After fixation in Davidson’s solution, liver tissues were embedded in paraffin wax and cut at 4 µm thickness. Tissues were antigen-retrieved by boiling in a buffered citrate solution (pH 6.0) for 20 min using the microwave after being deparaffinized and rehydrated. Slides were blocked using horse serum (10%) and BSA (1%) in PBS for 60 min at RT. Tissues were incubated overnight with a diluted blocking solution containing antibodies (anti-fibrinogen mouse monoclonal primary antibody, 1:150). Slides were washed in tween 20 (0.05%) in PBS and incubated with conjugated Cy3 anti-mouse secondary Ab (1:300 dilutions in blocking buffer) for 30 min. Slides were then incubated with DAPI (4,6-diamidino-2-phenylindole), and figures were captured and analyzed using a Leica DM 5500B epifluorescence microscope (Wetzlar, Germany). For measurement of the fluorescence intensity, at least 3 animal tissue samples were used from each group and 15 different field images from each section were recorded using Image-J/NIH software. Mean pixel intensity (from 0 to 255 scale values, next subtraction of the background) was used and calculated using GraphPad Prism 8.

Z-stack images were obtained using a Leica DM5500B epifluorescence microscope with a 63x oil objective (axial resolution 0.772 μm) with 1 μm axial steps. The images obtained were from 1600 × 1200 to 8696 × 7706 pixels in size with a field of view between 203 × 152 μm and 1103 × 977 μm. Image stacks were analyzed using the Fiji version of ImageJ (RRID: SCR_002285).