Ab28740

Paraffin-embedded lung tissue sections were deparaffinized in xylene and rehydrated in a graded ethanol series to PBS. Antigen retrieval was performed by pressure cooking in citrate buffer for 10 min. The sections were permeabilized by incubation with 0.3% Triton X-100 and blocked with 5% donkey serum albumin in a humidified chamber for 1h, and were immunostained with primary antibodies to Mxi1 (1:200, ab28740, Abcam Ltd., Cambridge, United Kingdom) or α-smooth muscle actin (α-SMA) (1:200, 48938, CST, Boston, United States). After overnight incubation, sections were washed and incubated with the respective secondary antibodies, donkey anti-rabbit IgG (1:1,000, Jackson Immuno, PA, United States), alexa 594 donkey anti-rabbit IgG (1:1,000, Jackson Immuno, PA, United States), or oralexa 488 donkey anti-mouse IgG (1:1,000, Jackson Immuno, PA, United States) for 1 h. For immunohistochemistry, sections were counterstained with hematoxylin and detected by incubation with the DAB substrate. For immunofluorescence staining, sections were counterstained with nuclear DAPI (1:1,000) and mounted with fluorescent mounting medium.

Human PASMCs grown on chamber slides were fixed with 4% paraformaldehyde, permeabilized by incubation with 0.3% Triton X-100 and blocked with 5% donkey serum albumin for 2 h. Then, slides were immunostained overnight with primary antibodies to Mxi1 (1:100, ab28740, Abcam Ltd., Cambridge, United Kingdom), followed by 1 h incubation with a secondary antibody, alexa 594 donkey anti-rabbit IgG (1:200, Jackson Immuno, PA, United States). After incubation, slides were counterstained with nuclear DAPI (1:1,000) and mounted with fluorescent mounting medium. Fluorescent images were taken with fluorescence microscope (Olympus, Japan).