Widefield microscope

For immunofluorescence, samples frozen in OCT were cut, fixed with cold acetone (Fisher Scientific) or 10% Neutral Buffered Formalin (Fisher Scientific), and incubated with primary antibodies as above overnight at 4°C. Subsequently they were incubated with Cy5-conjugated secondary antibodies (Jackson Immunoresearch, West Grove, PA) and counterstained with the nuclear stain 4',6-diamidino-2-phenylindole (DAPI; Invitrogen). Slides were imaged with a Zeiss wide field microscope, with 4–5 representative images taken per sample. For blood vessel staining, the percentage VE-cadherin positive area per image was calculated using Image J (NIH). When photos of stained tissue are shown in figures, they are representative images, with quantification values in the middle of the range.

Pictures of H&E-stained distal lungs were acquired using a Zeiss widefield microscope (AxioImager). Alveolar spaces, thickness of alveolar walls, and length of secondary septae were measured using ImageJ (https://imagej.nih.gov/ij/) according to the American Thoracic Society guidelines^{70 (link)}. A minimum of five representative, non-overlapping fields of view from lungs of at least three mice from each group were evaluated.

Cells were seeded in Lab-Tek glass-bottomed eight-chambered slide (155411, Thermo Scientific) and pretreated as indicated. The chambered slide was then placed on the contained stage of microscope with 5% CO₂ at 37 ℃. Cell motility was monitored with the widefield microscope (Zeiss, Germany). Cell positions were recorded at every 10-min interval over a period of 24 h and then processed with Track Object Tool in Metamorph analysis software.

The anti-senescence activity of the BNC-G-HDE2 membrane was evaluated using the β-galactosidase (β-gal) staining assay. The BNC-G membrane was used as control. To prepare the membranes extracts, sterilized samples (10 cm²) were incubated in 10 mL of complete DMEM medium as previously described for the cytotoxicity assay.
NIH/3T3 cells were seeded in 12-well plates at a density of 2.5 × 10⁴ cells/well and allowed to stabilize for 24 h. Subsequently, 12.5 µM etoposide was used to induce cellular senescence in NIH/3T3 for 24 h. Etoposide-stimulated cells were then treated with the BNC-G-HDE2 membrane extract for another 24 h. As control, non-treated cells were incubated with DMEM medium or with extracts of BNC-G and BNC-G-HDE membranes. After incubation, culture medium was removed, and cells were washed with PBS and marked with β-gal solution prepared as described by the manufacturer (Cell Signaling Technology, Danvers, MA, USA). The analysis of the positive cells for senescence was performed at 20× magnification using a widefield microscope (Carl Zeiss, Oberkochen, Germany). At least 3 independent experiments were performed in replicate, and the percentage of β-gal-positive cells was determined using four microscopic images.