Tissues were fixed by adapting a method previously used to preserve colonic mucus using methanol based Carnoy’s fixative (methacarn)43 (link). Methacarn is a highly permeant non-crosslinking preservative that rapidly penetrates tissues transmurally while minimizing mucus disruption and tissue shrinkage43 (link). Animals were carried through full dose response curves. During peak increases in RL following 10 mg/ml MCh, mice were rapidly euthanized by anesthetic overdose and cessation of mechanical ventilation. Mice remained connected to the ventilator, which by default stopped at end-expiration (3 cm H2O PEEP). Skin covering the lower sternum was removed, and 400 μl of methacarn was injected into the pleural space. The trachea was ligated immediately below the tracheal cannula. These steps took less than 15 seconds, thus minimizing changes in airway constriction and mucus transport. Lungs were fixed in situ for 30-60 min, then removed and immersion fixed with the trachea still ligated for 24-48 h in methacarn at 4°C. Tissues were transferred to 100% methanol (changed twice over a 48 h period). While remaining fully immersed in methanol, left lungs were sectioned into uniform 1.92 mm thick filets and imaged to calculate volume by Cavalieri estimation (319 ± 31 mm3 and 322 ± 24 mm3 for WT and Muc5ac−/− mice, respectively). Tissues were then paraffin processed and sectioned (5 μm thickness).
Histological assessments were made using AB-PAS and PAFS to assess total mucin, Muc5ac (clone 45M1, 1:100 dilution), and MUC5B (custom polyclonal antisera, 1:2,000) production37 (link),65 (link),66 (link). Intracellular mucin content levels and the extent of mucus plugging were examined by systematic uniform random sampling67 (link). Stained slides were scanned by two blinded investigators using an Olympus BX63 microscope and cellSens software (Olympus USA, Center Valley, PA). At least 5 axial bronchi per mouse were analyzed. For non-axial “bronchiolar” airways, samples were analyzed by selection of every fifth 20× image in a continuous x−/y− map of all lung samples per slide. Mucin and airway area were calculated using point-grid counting. Basement membrane length and airway circumferences were measured in cellSens. Data were calculated as volume densities (volume per unit surface area of basement membrane)9 (link),65 (link) and as extracellular mucus volume fractions relative to lung and airway volumes (multiplied by 100 to express as % occlusion)67 (link).
Histological assessments were made using AB-PAS and PAFS to assess total mucin, Muc5ac (clone 45M1, 1:100 dilution), and MUC5B (custom polyclonal antisera, 1:2,000) production37 (link),65 (link),66 (link). Intracellular mucin content levels and the extent of mucus plugging were examined by systematic uniform random sampling67 (link). Stained slides were scanned by two blinded investigators using an Olympus BX63 microscope and cellSens software (Olympus USA, Center Valley, PA). At least 5 axial bronchi per mouse were analyzed. For non-axial “bronchiolar” airways, samples were analyzed by selection of every fifth 20× image in a continuous x−/y− map of all lung samples per slide. Mucin and airway area were calculated using point-grid counting. Basement membrane length and airway circumferences were measured in cellSens. Data were calculated as volume densities (volume per unit surface area of basement membrane)9 (link),65 (link) and as extracellular mucus volume fractions relative to lung and airway volumes (multiplied by 100 to express as % occlusion)67 (link).
