Mouse anti α sma primary antibody

Image acquisition was performed with a Leica DMI6000B microscope equipped with the total internal reflection fluorescence (TIRF), Nomarski interference contrast (DIC) modules. LAS-AF deconvolution software was used for image processing. For phenotype plasticity L.t.EMT⁺RGM1, l.t.EMT⁻RGM1 and RGM1 cells were trypsinized and seeded at 8 × 10⁶ cells per well and each of them were cultured for 24 h in all 3 types of media: Hp-AGF supernatant, GF supernatant, and DMEM + 10% FBS. Then, the cells were photographed.
For the immunofluorescence, the α-SMA localization was analyzed in formaldehyde (3.7%)-fixed, Triton X-100 (0.1%) permeabilized cells. The mouse anti- α-SMA primary antibody (A2547, Sigma-Aldrich, Saint Louis, MO, USA) was used. Specimens were labeled with Alexa 488-conjugatedgoat anti-mouse IgG (No. A11001, Invitrogen, Carlsbad, CA, USA) and counterstained with Hoechst 33258 (No. B2883, Sigma-Aldrich, St. Louis, MO, USA). For morphological and fluorescent staining evaluation, at least 20 microscopic fields of view were analyzed.

Tissue sections were deparaffinized and rehydrated, followed by antigen retrieval with 0.8% SDS for 10 min. Blocking and permeabilization was done by incubating sections in 10% fetal bovine serum and 0.5% TritonX-100 for 30 min. Sections were incubated for 90 min with mouse anti-αSMA primary antibody (A2547; Sigma-Aldrich, St. Louis, MO, USA) and 60 min with secondary goat antibody conjugated with AF488 (ab150141; abcam, Cambridge, UK). In order to detect the reporter fluorescent protein tdTomato in the lung tissue, frozen sections were fixed in 4% PFA for 5 min. Both cell nuclei from paraffin and the frozen sections were counterstained with Roti-Mount FluorCare DAPI (Roth, Karlsruhe, Germany). All steps were performed at room temperature.