Ab69090

Tissue samples were homogenized in lysis buffer (50 mM of Tris–HCl, 150 mM of NaCl, 2 mM of EDTA, 2 mM of EGTA, 0.2% Triton X-100, 0.3% NP-40, 0.1 mM of PMSF, and 1 µg/ml of pepstatin A) and then separated by 10% SDS-PAGE under reducing conditions. After electrophoresis, samples were transferred to PVDF membranes (IPVH00010, Millipore, Bedford, MA, USA), blocked with 5% skimmed milk in TBS/0.5% v/v Tween 20 for 1 h, washed with TBS/Tween, and incubated with anti-HO-1 (1:2000 dilution, ADI-OSA-150-D Enzo Life Technologies), rabbit anti-ferritin light chain (1:500 dilution, ab69090, Abcam), rabbit anti-ferritin heavy chain (1:1000 dilution, Thermo Fisher 701934), and anti-phospho Nrf2 (1:1000 dilution, bs-2013R, Bioss). Antibodies were diluted in 5% milk TBS/Tween. Blots were washed with TBS/Tween and incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000, Amersham, Aylesbury, UK). After being washed with TBS/Tween, blots were developed with the chemiluminescence method (ECL Luminata Crescendo, WBLUR0500, Millipore) and scanned using the ImageQuant LAS-4000 (GE Healthcare). Blots were then probed with mouse monoclonal anti-α-tubulin antibody (1:5000, T6199, Sigma, MO, USA), and levels of expression were corrected for minor differences in loading. Quantification was expressed as arbitrary densitometric units (AU).

Fresh tracheal cryosections from mice infected with Af-GFP were fixed with 4% paraformaldehyde in PBS pH 7.4 for 15 min at room temperature. Tissue was then permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature. Then, tracheas were then incubated with 1% BSA, 22.52 mg/mL glycine in PBST (PBS + 0.1% Tween 20) for 30 min to block unspecific binding of the antibodies. Tissue was incubated with primary antibody ferritin (ab69090; Abcam, Cambridge, UK) in 1% BSA in PBST for 1 h at room temperature. Following the tissue was incubated with secondary antibody goat anti-Rabbit IgG Texas Red (ab6719; Abcam, Cambridge, UK) in 1% BSA for 1 h at room temperature in the dark. Tracheas were then mounted using ProLong Gold (P10144; Invitrogen^TM,,Waltham, MA, USA). Images and z stacks were taken using a confocal microscope (Leica Stellaris 8). Fluorescence was quantified by ImageJ.