NMJ denervation was detected according to the previously described protocol [22 (link)]. Briefly, tibialis anterior muscles (TAM) were dissected from mice transcardially perfused with PBS under deep anesthesia and snap-frozen in isopentane cooled on dry ice. Serial 20 µm cryostat longitudinal muscle sections were collected on poly-Lysine objective slides (VWR International), fixed in chilled acetone for 10 min, incubated in a blocking solution (0.3% Triton, 10% NGS in 0.01 M PBS) for 1 h at 22 °C and left overnight at 4 °C with anti-SV2 primary antibody (1:100, DSHB) in 0.15% Triton, 5% NGS, 0.01 M PBS. Then the sections were incubated with goat anti-rabbit 647 (1:500, Alexa Fluor® Dyes, Life Technologies) secondary antibody and with bungarotoxin (1:500, Invitrogen) conjugated with Alexa Fluor® 488 (Life Technologies). Innervated neuromuscular junctions were identified by the bungarotoxin labelling, totally or partially co-localized with synaptophysin labelling. Endplates marked with bungarotoxin only were considered denervated. Data were expressed as the percentage of the denervated plaques over the total ones counted in 8 adjacent frames per section. Five sections at 20× magnification were analyzed for each mouse. Fluorescence images along the z axis were taken by Olympus confocal microscopy using a 20× objective and z-stacking was performed by using ImageJ software.
Poly lysine objective slides
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Poly-lysine objective slides are a type of microscope slide coated with a positively charged polymer, poly-L-lysine, to enhance cell adhesion. These slides provide a surface that promotes the attachment and growth of cells during microscopic observation and analysis.
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Lab products found in correlation
Vs110, by Olympus (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Alexa fluor dye, by Thermo Fisher Scientific (1 mentions)
Olyvia, by Olympus (1 mentions)
Alexa fluor 594, by Thermo Fisher Scientific (1 mentions)
Confocal microscopy, by Olympus (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
Fv500, by Olympus (1 mentions)
Hoechst, by Roche (1 mentions)
Anti ki67, by Abcam (1 mentions)
6 protocols using poly lysine objective slides
1
Quantifying Neuromuscular Junction Denervation
2
Neuromuscular Junction Innervation Analysis
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Tibialis anterior (TA) was dissected out, and snap-frozen in isopentane cooled in liquid nitrogen. 20-μm serial longitudinal cryosections were collected on poly-lysine objective slides (VWR International). Five serial sections (average ~ 70 NMJs) per animal were analysed. Muscle sections were stained with anti-synaptic vesicle protein (SV2; 1:100; Developmental Studies Hybridoma Bank), mouse anti-neurofilament 165 kDa (2H3; 1:50; Developmental Studies Hybridoma Bank), followed by 647 anti-mouse secondary antibody (1:500; Invitrogen). α-Bungarotoxin coupled to Alexa Fluor 488 (1:500) (Invitrogen) was then added and left for 2 h at room temperature. Innervation analysis was performed directly. Images of all genotypes for the innervation analysis were obtained with an Olympus virtual slide system VS110 (Olympus, centre Valley, PA, USA) at 40X magnification. The percentage of neuromuscular innervation was quantified in OlyVIA (Olympus) based on the overlay between neurofilament (SV2/2H3) staining and α-BTX labelled endplates. Endplates were quantified as occupied when any neurofilaments staining were overlying the endplate and as vacant when there was no overlay. The endplate area was determined using Fiji software (ImageJ, National Institutes of Health).
3
Neuromuscular Junction Visualization
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Tibialis anterior muscles were dissected out and snap-frozen in isopentane cooled in liquid nitrogen. Twenty-micrometer serial longitudinal cryosections were collected on poly-lysine objective slides (VWR International). The sections were stained with anti-synaptophysin (SYP) (1:100; Synaptic System) as pre-synaptic marker. The secondary antibody was FITC-labeled goat anti-rabbit (1:500, Invitrogen). Subsequently, the sections were stained with α-bungarotoxin (α-BTX) coupled to Alexa Fluor 594 (1:1200; Invitrogen) as post synaptic marker. Fluorescence images along the z-axis were taken by Olympus confocal microscopy using a ×20 objective, and z-stacking was done using Fiji software (with Z-Stack Projection/SUM command) (ImageJ, US National Institutes of Health, Bethesda, MD, USA). End plates were scored as innervated if there was complete overlap between SYP and a-BTX staining and scored as denervated if they showed only α-bungarotoxin staining.
4
Nerve Dissection and Imaging Protocol
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After 0.1 M PBS perfusion, radial and sciatic nerves were dissected out from the same animal and mounted in OCT. Serial longitudinal sections (14 μm) were collected on poly-lysine objective slides (VWR International). For each slice, fluorescence fields were taken the laser scanning confocal microscope (Olympus, Tokyo, Japan). The mean grey value of the immunoreactivity was assessed through Fiji (Image J, U. S. National Institutes of Health, Bethesda, Maryland, USA) for each section in the analysis.
5
Quantifying Neuromuscular Junction Innervation
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TA muscles were dissected out and snap‐frozen in liquid nitrogen. Serial longitudinal cryosections (20 μm) were collected on poly‐lysine objective slides (VWR International). Six serial sections per animal were analyzed. Muscle sections were stained with anti‐synaptic vesicle protein (SV2; 1:100 Developmental Studies Hybridoma Bank) and mouse anti‐neurofilaments (2H3 1:50 Developmental Studies Hybridoma Bank). α‐bungarotoxin coupled to Alexa Fluor 488 (1:500 Invitrogen) was then added and left for 1 h at room temperature. Images were obtained with an Olympus virtual slide system VS110 (Olympus, Center Valley, USA) at 20×‐magnification. The colocalization channel between neurofilament and BTX immunostaining was produced for each Z‐stack. The percentage of neuromuscular innervation was quantified in OlyVIA based on the overlap between neurofilaments (SV2/2H3) staining and a‐BTX labeled endplates.
NMJ morphology was evaluated using the “NMJ‐morph” workflow in Fiji (Image J, U.S. National Institute of Health, Bethesda, Maryland, USA) as previously described15 . The images of α‐BTX+ endplates were acquired under a sequential scanning mode on an Olympus confocal scan unit FV500 (Olympus, Center Valley, USA) with the following parameters: 8 bit depth, 512 × 512 frame size, ×60 magnification, ×2.5 zoom and 1 μm Z‐stack interval.
NMJ morphology was evaluated using the “NMJ‐morph” workflow in Fiji (Image J, U.S. National Institute of Health, Bethesda, Maryland, USA) as previously described
6
Lung Tissue Analysis in Anesthetized Mice
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Mice were deeply anesthetized with Medetomidine, 2 mg kg−1, and Ketamin, 150 mg kg−1, and transcardially perfused with 50 mL of 0.1 M PBS. The lungs were harvested and snap-frozen in liquid nitrogen; 20 μm longitudinal serial lung cryosections were collected on polylysine objective slides (VWR International). Sections were fixed in cold acetone for 10 minutes, then blocked in PBS with 10% normal goat serum and 0.3% Triton X-100, for 1 hour at room temperature. Sections were incubated overnight with primary antibodies: anti-CD11b, rat (1:200; BioRad) or anti-Ki67, rabbit (1:200; Abcam) at 4°C, then stained with the secondary antibody Alexa 488 anti-rat or anti-rabbit. The nuclei were counterstained whit Hoechst (1:1000; Roche). Images were acquired using a sequential scanning mode with an A1 Nikon confocal running NIS Elements at 20X or 40X magnification. Image analyses were done as detailed in Supplemental Methods.
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