Immunofluorescence Imaging of Lung Signaling

Tissues were processed as described above. Immunofluorescence studies were done according to a recently published study^{28 (link)}. In brief, lung sections were then incubated in blocking solution (5% bovine serum albumin (BSA) + 0.3% Triton X-100 in PBS) for 1 h, followed by incubation overnight at 4 °C with primary antibodies [pEGFR, pERK1/2, pAkt, pI-κB, (1:50 - 1:100 dilution); Cell Signaling, USA], diluted in 1% blocking solution. Subsequently, sections were washed and incubated with secondary antibody conjugated to Alexa Fluor 555 (Goat anti rabbit SFX kit; Life Technologies, USA, 1:400 dilution) for 2 h at room temperature in the dark. pI-κB was measured as a surrogate marker for NF-κB activation. After washes in PBS sections were stained with 4’,6 diamidino-2- phenylindole and mounted. Images were captured on a ZEISS LSM 700 confocal microscope and fluorescence intensity estimated in defined fields using Image J software package.