Pen membrane slides 2

LCM of motor neurons was carried out essentially as described previously (Lotti et al. 2012 (link)). Briefly, to retrogradely label motor neurons, the IL and QL muscles of P2 wild-type and SMA mice were exposed, and ∼1 µL of CTb conjugated to Alexa 488 was delivered by intramuscular injection using a finely pulled glass microelectrode. At P6, the spinal cord was dissected, and the L1–L3 segments were embedded in OCT and flash-frozen. Cryosections of 14 µm were mounted on PEN-membrane slides 2.0 (Zeiss) and fixed in 100% ethanol for 15 sec prior to LCM. Following air drying for 30 sec, individual motor neurons were microdissected using a DM6000B microscope equipped with a LMD6000 laser capture unit (Leica). Approximately 200 motor neurons were collected bilaterally from L1–L3 spinal segments of one or more mice for each sample group and biological replicate.

Isolation of motor neurons by laser capture microdissection was performed as previously described (Van Alstyne et al., 2018 (link); Lotti et al., 2012 (link)). Briefly, Cholera toxin B (CTB) conjugated to Alexa 488 was delivered by intramuscular injection in the iliopsoas and QL muscles of wild type and SMA mice at P2 using a finely pulled glass microelectrode. The L1-L3 spinal segments were dissected from the injected mice at P6, embedded in OCT, and flash frozen prior to sectioning with a cryostat. Sections (14 μm) were mounted on PEN-membrane slides 2.0 (Zeiss), fixed in 100% ethanol for 15 s, and air dried for 30 s prior to microdissection of CTB⁺ motor neuron somata using a DM6000B microscope equipped with a LMD6000 laser capture unit (Leica).