Ab72996

Gactrocnemius muscle was excised, dissected into smaller sections, and fixed in 4% paraformaldehyde, and then incubated with 1 μg/mL Rhodamine Red-Conjugated Bungarotoxin (Sigma-Aldrich). Tissues were then treated with methanol at −20 °C for 5 min, and afterwards blocked in blocking solution (1 mg/mL BSA, 0.1% triton in PBS) for 1 hour. Tissues were then rocked with appropriate primary antibodies diluted in blocking solution at room temperature overnight. Antibodies were used at the following concentrations: NFH 1:500 (Chicken; Abcam, ab72996); Elavl2 1:50 (Rabbit; Proteintech 14008-1-AP); Synaptophysin 1:500 (Mouse; millipore, mab5258) After washing, secondary antibodies (DyLight 405 anti-chicken 1:500; AlexaFluor 488 anti-Rabbit 1:500; AlexaFluor 647 anti-mouse 1:500) were added for 4 hours at room temperature. Muscle fibers were spread into monolayers under a stereomicroscope and affixed to slides using VectaShield (Vector Laboratories).

Sensory neurons from WT and Isl1-Dync1h1^+/− mice were grown on glass coverslips coated with poly-L-lysine and laminin for 24 or 48 h and then fixed using 4% paraformaldehyde (PFA) in 1× PBS. Blocking and permeabilization was done with 10% donkey serum and 0.2% Triton X-100 in PBS for 1 h. Coverslips with neurons were incubated overnight at 4°C with chicken anti-NFH (Abcam, ab72996, 1:2000) and rabbit anti-Dync1h1 (Proteintech, 12345-1-AP, 1:500). The next day, they were washed three times in 1× PBS and incubated for 1 h with donkey anti-chicken-IgY conjugated to Alexa Fluor 488, donkey anti-rabbit-IgG conjugated to Alexa Fluor 647 (1:1000; Jackson ImmunoResearch) and DAPI (Abcam, ab228549, 1:5000). After three washes in 1× PBS, coverslips were rinsed in ddH₂O and mounted with Fluoromount-G^TM.

Culture of sensory neurons from DRG were performed as previously described (Terenzio et al., 2018b (link)). Adult DRG were dissociated by enzymatic treatment (papain and a mix of collagenase/dispase), triturated in Hanks’ balanced salt solution (HBSS)-supplemented with 10 mM glucose and 5 mM HEPES pH 7.35, and recovered by centrifugation (1000 g for 8 min) through 20% Percoll. Sensory neurons obtained from this procedure were then subjected to immunostaining. Primary antibodies used in this study are the following: chicken anti-NFH (Abcam, ab72996, 1:2000), rabbit anti-Dync1h1 (Proteintech, 12345-1-AP, 1:500), mouse anti-importin β1 (monoclonal, generated in-house), rabbit anti β-III tubulin (Abcam, ab18207, 1:2000), recombinant rabbit monoclonal anti-ATF3 (Abcam, ab207434, 1:1000, for IF) and rabbit polyclonal anti-Stathmin2/Scg10 (Novus, NBP1-49461, 1:1000, for immunofluorescence). Secondary antibodies used for immunostaining are anti-chicken-IgY, -mouse-IgG or -rabbit-IgG conjugated to Alexa Fluor 488, 594 or 647 (Jackson ImmunoResearch, 1:1000). Secondaries for immunoblotting were HRP-conjugated anti-rabbit-IgG antibodies (Bio-Rad Laboratories, 1:10,000).