Prolong gold antifade reagent with 6 diamidino 2 phenylindole dapi

A549 cells were inoculated on glass coverslips and transfected with indicated DNA for 24 h. The transfected cells were fixed with 4% paraformaldehyde in PBS for 5 min and permeabilized with 0.2% Triton X-100, 0.04% SDS in PBS for 5 min. After blocking in 10% normal goat serum (Boster, Wuhan, China) at room temperature for 30 min, cells were incubated overnight at 4 °C with primary antibody against Flag epitope or NCL. The appropriate Alexa-Fluor-coupled secondary antibody (Invitrogen; 1:750) was applied for 1 h at room temperature. The cells were mounted in ProLong Gold antifade reagent with 6-diamidino-2-phenylindole (DAPI) (Life Technologies) and analyzed under an Olympus FV1000 laser confocal microscope (Olympus, Japan).

For immunofluorescence, cultured MSCs were fixed in 4% paraformaldehyde (PFA) and processed as described previously^{58 (link)}. Cells were stained with primary and secondary antibodies listed in Table S1. For immunohistolocalization assays, freshly collected fetal SGs and adult SMGs were fixed and embedded as described previously^{59 (link)}. 10 µm cryostat sections were subsequently stained with primary and secondary antibodies (Table S1) and were counterstained with ProLong® Gold antifade reagent with 6-diamidino-2-phenylindole (DAPI) (Life Technologies, Grand Island, NY). Images were captured on Leica CTR6500 (Leica Microsystems, Buffalo Grove, IL) and EVOS epifluorescence microscopes (Thermo Fisher Scientific, Waltham, MA). Images were further analyzed using NIH-ImageJ software (NIH, Bethedsa, Maryland). Quantification of the images was done using NIH-ImageJ software to analyze 10 to 15 random images at 20x magnification from ≥3 fetal and adult SMG sections.