Lsm 510 inverted confocal microscope

Fresh frozen muscle sections from Patient 1 were labelled with Alexa Fluor® 594 conjugated α-bungarotoxin (Life Technologies, Cat. No. B13423) and Alexa Fluor® 488-fasciculin (Life Technologies, special order) at 1 μg/ml for 1 h at 37°C to stain for AChRs and acetylcholinesterase (AChE), respectively. The presynaptic Schwann cell marker S100β was labelled using a mouse monoclonal anti-S100β antibody (Sigma, Cat. No. SAB1402349) and the corresponding fluorescently conjugated secondary antibody (Life Technologies, Cat. No. R37115). Then, sections were washed in PBS and fixed for 10 min in 3% paraformaldehyde at room temperature. Images from the muscle endplates were taken using a Zeiss LSM 510 inverted confocal microscope. Co-localization studies were performed using ImageJ software (Schindelin et al., 2012 (link)).

Third-instar WCS and homozygous spastin^5.75 or trans-heterozygous flower^DB25/DB56 larvae were filleted, dissected and immunostained using standard methods (e.g. Ozdowski et al., 2011 (link)). Briefly, larvae were dissected in room temperature PBS and fixed for 30 minutes in 4% paraformaldehyde, immunostained at 4°C overnight using the neuronal membrane marker rabbit anti-HRP (1:100; Jackson ImmunoResearch, PA, 323-005-021) alone or with mAb 22C10 to label microtubules (mouse anti-Futsch, 1:50; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA). Secondary antibodies (Alexa Fluor 488 goat anti-rabbit A-11070 and Alexa Fluor 568 goat anti-mouse A-11031; 1:400; Life Technologies, Grand Island, NY) were incubated for 2–3 hours at room temperature. Fillets were mounted in H-1000 (Vector Laboratories, Burlingame, CA) and z-series images of muscle 4 synapses from larval segments 2–4 acquired on a Zeiss LSM 510 inverted confocal microscope using 63× 1.4 N.A. or 100× 1.2 N.A. PlanApo objectives (Oberkochen, Germany).

Paraffin sections were de-waxed and incubated in primary antibody overnight at 4°C. Primary antibodies were detected using the Vectastain Elite ABC kit (Vector Labs, UK) and diaminobenzidine detection (BD Bioscience, UK) following the manufacturers' instructions. Antigen retrieval was performed for Vangl2, phospho-histone H3 and CC10 staining using citrate buffer (pH 6) incubation in a microwave for 3×3 min. Cryosections were incubated with Rhodamine-conjugated Phalloidin (Biotium, Hayward, CA, 00027; 1:200) following the manufacturer's instructions.
Lung slices were fixed in 4% paraformaldehyde (PFA) for 15 min, and permeabilized in 0.5% Triton X-100 for 30 min. Non-specific protein binding was blocked for 1 h at room temperature using 1% BSA and 0.2% Triton X-100 in PBS (PBS-BT). Slices were incubated with primary antibody overnight at 4°C, washed several times in PBS then incubated with donkey anti-rabbit-conjugated Alexa Fluor 594 (Life Technologies, A21207; 1:500) or goat anti-Armenian-Hamster-conjugated Cy3 (Jackson ImmunoResearch, West Grove, PA, 127-165-099; 1:500) secondary antibodies, and imaged using a Zeiss LSM-510 inverted confocal microscope. Controls, where the primary antibody was omitted, were included in every immunostaining experiment.

To measure cytosolic and mitochondrial ROS production in HPASMCs, cytosolic specific staining with DCFH and DHE and mitochondrial specific staining with Mitosox were used [18 (link)]. HPASMCs were incubated in 24-well plates and treated with different reagents for 4 h. Then washed the cells with PBS and incubated with pre-warmed PBS containing at a final working concentration of 10 μM DCFH dye for cytosolic H₂O₂ detection at 37 °C for 30 min and 10 μM DHE fluorescent probe for cytosolic superoxide anion (O²⁻) at 37 °C for 1 h. The same process for mitochondrial ROS detection, except incubation in Mitosox at 200 nM. The fluorescence of DCFH was measured using excitation and emission wavelengths of 480 and 535 nm, and the fluorescence of DHE was measured using excitation 510–560 nm and emission of 590 nm, respectively. MitoSOX Red fluorescence was measured at 583 nm following excitation at 488 nm using a Zeiss LSM 510 inverted confocal microscope. Thirty smooth muscle cells in each group were randomly photographed, and their total fluorescence was recorded. Data were analyzed using Flow Version software.