Hpds o ir pulsed laser

Tracheal explants from mice expressing membrane tomato under the ROSA promoter (stock no. 007676) were euthanized and dissected as described above. Tracheal samples were bisected as described with one half placed in DMEM/F12 media, while the other half was placed into DMEM/F12 with 10 µM of methacholine (dry powder dissolved in DMEM/F12, Sigma A2251). After 30 min, 1 hr, 1.5 hr, and 2 hr of incubation, the samples were fixed in 4% PFA in PBS for 20 min at room temperature with agitation. Samples were then washed with PBS three times for a total of 1 hr. Samples were permeabilized in PBS-0.3% Triton X-100 (PBST) for 30 min with gentle agitation. The samples were stained with CC10 primary antibody (aka Scgb1a1, 1:500; SC-9772, Santa Cruz) at 37°C for 2 hr, diluted in 1% BSA-0.3% PBST. Samples were rinsed in PBS for 30 min and incubated with secondary antibody for 30 min with a subsequent 30 min PBS wash. As above, imaging was conducted on the Olympus FVMPE-RS multiphoton laser scanning microscope equipped with a MaiTai HPDS-O IR pulsed laser (900 nm for AF488) and an INSIGHT X3-OL IR pulsed laser (1050 nm for membrane tomato). Laser power was kept constant for all images, and all tracheal samples were acquired using the same settings. ImageJ was used for quantifying fluorescence intensity on maximal projection images.

Cd11c-mCherry mice were stimulated with LPS (30 µg in 40 µL PBS, delivered intranasally as a single dose, 5 days prior to experimentation). Explants from these mice were then cultured in DMEM/F12 containing 1 mg/ml 10Kd FITC-dextran (Sigma FD10S) and 10 µM of methacholine (Sigma A2251). Imaging was conducted on the Olympus FVMPE-RS multiphoton laser scanning microscope equipped with a MaiTai HPDS-O IR pulsed laser (800 nm for FITC) and INSIGHT X3-OL IR pulsed laser (1050 nm for mCherry) with 1024×1024 scan size and a step size 0.5 µm. A 15× optical zoom and line averaging scan were used for high-resolution imaging.

Explants were incubated with 10Kd FITC-dextran (Sigma FD10S) diluted into DMEM/F12 media at a concentration of 1 mg/ml with 10 µM methacholine (dry powder dissolved in DMEM/F12, Sigma A2251). Alternatively, explants were treated with FITC-ovalbumin (Thermo O23020) diluted into DMEM/F12 media at a concentration of 0.1 mg/ml with 10 µM methacholine (dry powder dissolved in DMEM/F12, Sigma A2251). Imaging was conducted on the Olympus FVMPE-RS multiphoton laser scanning microscope equipped with a MaiTai HPDS-O IR pulsed laser (800 nm for FITC) and an INSIGHT X3-OL IR pulsed laser (1050 nm for Draq5 or membrane tomato) with 2× optical zoom, 1024×1024 scan size, and a step size of 0.5 µm.