Cf640r

Antibodies against p80 (62 (link)) were from the Geneva Antibody Facility (University of Geneva, Switzerland); anti-Ub (FK2) monoclonal antibodies were from Enzo Life Sciences. The mCherry fluorescent signal was enhanced with rat monoclonal (MAb) anti-red fluorescent protein (anti-RFP) antibodies (Chromotek). As secondary antibodies, goat anti-rabbit, anti-mouse, and anti-rat IgG coupled to Alexa 488 or Alexa 546 (Thermo Fisher Scientific) or CF640R (Biotium) were used. Cells were fixed with cold methanol (MeOH) as described previously (40 (link)). Images were recorded with Zeiss LSM700 and LSM800 confocal microscopes and a 63×/1.4-numerical-aperture (NA) or a 100×/1.4-NA oil-immersion objective. Fluorescent proteins or secondary antibodies were excited using the 405-nm (DAPI), 488-nm (GFP and Alexa 488), 555-nm (Alexa 546), and 639-nm (CF640R) laser lines.

At P3–P6, pups injected with fluorescent tracers on P0 were anesthetized by isoflurane and killed by rapid cervical dislocation. Brains were submerged in 4°C, continuously 100% O₂-saturated buffer: 140 mm NaCl, 5 mm KCl, 2 mMCaCl₂, 5 mm glucose, and 10 mm HEPES. The brains were oriented for vibratome sectioning with caudal ends up and rostral sides attached to an agar block perpendicular to the blade. A single 700-μm slice of the medulla was prepared containing the fundamental respiratory control circuit: hypoglossal motor nucleus and rootlets, intermediate reticular formation interneurons, and pre-Bötzinger complex (Smith et al., 1991 (link)). Slices were incubated in NMDG recovery solution: 93 mm NMDG, 93 mm HCl, 2.5 mm KCl, 1.2 mm NaH₂PO₄, 25 mm NaHCO₃, 20 mm HEPES, 25 mm D-glucose, 10 mm MgSO₄, and 0.5 mm CaCl₂ saturated by 95% O₂/5% CO₂ at 34°C for 10 min, then moved to a recording chamber (10 ml), perfused with room temperature artificial CSF (aCSF):125 mm NaCl, 3 mm KCl, 2 mm CaCl₂, 26 mm NaHCO₃, 5 mm glucose, and 5 mm HEPES equilibrated with 95% O₂ and 5% CO₂, pH 7.4 (5–10 ml/min; 24–25°C). In a subset of slices, intracellular injections of a highly photostable far-red (excitation laser 640 nm) biocytin fluorophore conjugated dye, CF640R were made (2%; Biotium) to visualize cellular and dendritic morphology.