Protein extraction from formalin-fixed paraffin-embedded
(FFPE) lung tissues and western blot analysis:
FFPE lung tissues (10 μm sections) from non-COVID-19 and COVID-19
patients were transferred into 1.5 ml centrifuge tubes, dewaxed in
xylene for 10 min and centrifuged. T issue pellets were washed 3x with
xylene and rehydrated with graded concentrations of ethanol (100 %, 85
% and 75 % for 2 min in each). After the ethanol wash, proteins in the
tissue pellets were solubilized in Tris-SDS buffer, pH 8.0, by
homogenization and sonication. Tissue homogenates were centrifuged at
10,000 g for 20 min and the supernatants were measured for total protein
concentrations. Equal amounts of proteins were resolved by 12% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer
onto nitrocellulose membrane. Membranes were then blocked with 5%
bovine serum albumin and incubated with anti-elastin (1:1000 dilution)
and anti-histone H3 Pan antibodies (loading control, 1:4000 dilution)
overnight at 4 0C. Next day, membranes were washed in
TBST and incubated with IRDye 800CW and IRDye 600CW conjugated secondary
antibodies. The membranes were washed 3 times and signal was measured
using the Odyssey Infrared Imaging System (LI-COR Biosciences).