Anti rabbit af647

All primary antibodies were incubated at 4°C overnight. In cases where rAAV‐mediated expression of GFP was restricted to 7 days, the GFP signal was amplified using an anti‐GFP antibody (A11122; Life Technologies) followed by a Goat Anti‐Rabbit‐488 secondary antibody (Life Technologies; both 1:500). In order to characterizes contacts onto interneurons, we stained with Rabbit anti‐vGlut1 (1:500; Synaptic Systems, #135303), Rabbit anti‐vGlut1/2 (1:500; Synaptic Systems, #135503), Guinea pig anti‐vGAT (1:1,000; Synaptic Systems, #131308), rabbit anti‐Synapsin (1:500; Millipore), and mouse anti‐Basson (1:200; ENZO) antibodies. In order to label moto neurons, we co‐stained with rabbit anti‐ChAT antibody (1:100; Abcam, #ab178850) and NeuroTrace435 (1:200; ThermoFisher, #N21479). Primary antibodies were diluted in 0.3% Triton 20 and PBS. All secondary antibodies were kept for 2 h at room temperature with gentle shaking. Secondary antibodies used are as follows: anti‐rabbit AF647 (1:500; ThermoFisher, #A32795), anti‐guinea pig Cy3 (1:500; Jackson ImmunoResearch, #706‐165‐148), anti‐guinea pig AF633 (1:500; Sigma Aldrich, #SAB4600129).

1˚: anti-pVI (a kind gift from Urs Greber (Institute of Molecular Life Sciences, Zurich, Switzerland)), affinity purified rabbit polyclonal, 1:8, staining technique: cover slip inversion). 2˚: anti-rabbit-AF647 (Thermo Fisher, 1:500).

Multiplexed imaging of distal lung was adapted from McCowan et al.^{82 (link)}. Antigen retrieval was performed on histological sections using Target Retrieval Solution, pH 6.0 (Agilent) (20 min at 97 °C) on a Dako PT retrieval module. Samples were permeabilized and blocked for 20 min in PBS/Neutral goat serum (NGS) 10%/BSA1%/TritonX-100 (Tx100) 0.3%/Azide 0.05% at 37 °C and stained with 150 μl rabbit anti-Keratin 5 (Polyclonal, Poly19055, BioLegend; 1:200) diluted in PBS/ NGS10%/BSA1%/ TX-100 0.3%/Azide 0.05% for 1 h. Samples were washed 3 times with PBS/BSA1%/TX-100 0.1%/Azide 0.05% before adding 150 μl of_a solution containing, anti-Histone H3 (C-terminus) AF 594 (BioLegend; 1:200), aSMA-Cy3 (clone 1A4, Sigma; 1:1000) and anti-rabbit-AF647 (polyclonal, A-21244, ThermoFisher) diluted in PBS/ NGS10%/BSA1%/ TX-100 0.3%/Azide 0.05% for 1 h. Samples were washed 3 times with PBS/BSA1%/TX-100 0.1%/Azide 0.05% and 2 times in PBS. Finally slides were mounted with Vectashield (Vector Laboratories, #H-1000-10).