The iDISCO(ace) protocol was developed as a modification of the original iDISCO+ method (19). This procedure enables the whole-tissue immunolabeling and 3D assessment of intended cellular structures, including but not limited to neural innervations, neuroendocrine cells, lymphatic vessels, and different immune cells, in the unsectioned lung tissues. The anesthetized mice were perfused sequentially with 20 ml of phosphate-buffered saline (PBS)/heparin (100 μg/ml) and 20 ml of PBS/1% paraformaldehyde (PFA)/10% sucrose/heparin (100 μg/ml). The lung tissues were dissected out and postfixed in PBS/0.5% PFA at room temperature for 2 hours. The tissues were washed with PBS at room temperature for 30 min twice. All the incubation steps were performed with gentle rotating.

The lung tissues were treated at room temperature with 25% acetone (diluted in ddH2O) for 1 hour, 50% acetone for 3 hours, and 25% acetone for 1 hour. The tissues were washed with PBS at room temperature for 30 min twice, followed by PBS/30% sucrose for 2 hours. The tissues were decolorized in PBS/30% sucrose/1% H2O2/10 mM EDTA-Na (pH 8.0) at 4°C overnight. The tissues were then washed with PBS at room temperature for 30 min twice, followed by PBS/0.2% Triton X-100/0.1% deoxycholate/10% DMSO/10 mM EDTA (pH 8.0) overnight. All the incubation steps were performed with gentle rotating.

Next, the tissues were blocked with PBS/0.2% Triton X-100/10% DMSO/5% normal donkey serum at room temperature overnight and then immunolabeled with intended primary antibodies (1:500 dilution) in PBS/0.1% Tween 20/heparin (10 μg/ml)/5% normal donkey serum at room temperature for 48 hours. The primary antibodies used in this study were rabbit anti-synaptophysin (Thermo Fisher Scientific, catalog no. 180130, RRID:AB_10836766), rabbit anti-Tuj1 (Covance, catalog no. MRB-435P, RRID:AB_663339), rabbit anti-PGP9.5 (ProteinTech Group, catalog no. 14730-1-AP, RRID:AB_2210497), rabbit anti-TH (Millipore, catalog no. AB152, RRID:AB_390204), goat anti-VChAT (Millipore, catalog no. ABN100, RRID:AB_2630394), goat anti-CGRP (Abcam, catalog no. ab36001, RRID:AB_725807), rat anti-LYVE1 (Thermo Fisher Scientific, catalog no. 14-0443-82, RRID:AB_1633414), rat anti-CD3 (BioLegend, catalog no. 100202, RRID:AB_312659), rat anti-CD11b (BioLegend, catalog no. 101202, RRID:AB_312785), rat anti–Siglec-F (BD Biosciences, catalog no. 552125, RRID:AB_394340), rat anti–Ly-6G (Thermo Fisher Scientific, catalog no. 16-9668-85, RRID:AB_2573127), and rat anti-F4/80 (BioLegend, catalog no. 123102, RRID:AB_893506). The tissues were washed with PBS/0.1% Tween 20/heparin (10 μg/ml) at room temperature for 12 hours, with the fresh buffer changed every 2 hours. The tissues were further immunolabeled with the corresponding Alexa Fluor dye–conjugated secondary antibodies (1:500 dilution; Thermo Fisher Scientific) in PBS/0.1% Tween 20/heparin (10 μg/ml)/5% normal donkey serum at room temperature for 48 hours. The tissues were washed with PBS/0.1% Tween 20/heparin (10 μg/ml) at room temperature for 24 hours, with the fresh buffer changed every 6 hours. All the incubation steps were performed with gentle rotating.

The immunolabeled lung tissues were washed with PBS at room temperature for 2 hours and embedded in PBS/0.8% agarose before the optical clearing. The tissue blocks were incubated at room temperature with 20% methanol (diluted in ddH2O) for 1 hour three times, 40% methanol for 1 hour, 60% methanol for 1 hour, 80% methanol for 1 hour, 100% methanol for 1 hour, and 100% methanol overnight. The tissue blocks were then incubated at room temperature with the mixture of dichloromethane and methanol (2:1) for 3 hours, followed by 100% dichloromethane for 30 min three times. The tissue blocks were lastly incubated at room temperature with 100% dibenzyl ether for 12 hours twice. All the incubation steps were performed with gentle rotating.

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