Fluorophore conjugated secondary antibody

Mice were anesthetized with sevoflurane and then sacrificed using infusion of saline solution followed by 4% paraformaldehyde (PFA). Harvested brains were post-fixed in 4% PFA for 24 h, then cryoprotected in 30% sucrose for 48 h at 4°C. Coronal brain sections (30-μM thickness) were cut using a cryostat microtome. Brain sections were blocked with 5% bovine serum albumin for 1 h at room temperature, then incubated with goat anti-ionized calcium binding adapter molecule 1 (Iba1) (#ab5076, 1:200, abcam, Cambridge, MA, USA), rabbit anti-glia fibrillary acidic protein (GFAP) (#ab7260, 1:1000, abcam, Cambridge, MA, USA), rabbit anti-Synaptophysin (#A6344, 1:100, abclonal, Wuhan, China), and rabbit anti-lysosomal associated membrane protein 1 (LAMP1) (#ab24170, 1:200, abcam, Cambridge, MA, USA) at 4°C overnight. After washing, brain sections were incubated with fluorophore-conjugated secondary antibodies (1:500, abcam, Cambridge, MA, USA) for 1 h at room temperature. Images were visualized using the Leica TCS SP8 STED 3× confocal microscope (Leica, Wetzlar, Germany). Laser power and gain were consistent across different experiments. Quantitative analyses were performed using Fiji software.

Cells were seeded onto sterile coverslips in plates at 60–70% confluence. The next day, the cells were washed once with PBS, fixed in 4% paraformaldehyde for 20 min and permeabilized with 0.1% Triton X-100 for 6 min. After blocking for 1 h with blocking buffer (1% bovine serum albumin in PBS buffer, pH 7.4), the cells were incubated with antibodies against to Smad2, pSmad2, E-cadherin, N-cadherin, LC3B, or SQSTM1/p62 overnight, followed by incubation with a fluorophore-conjugated secondary antibody (Abcam) for an additional 2 h. Cell nuclei were stained with 4′,6 diamidino-2-phenylindole (DAPI) for 1 min. A confocal laser-scanning microscope (TCS SP8; Leica, Wetzlar, Germany) was used to collect fluorescence images. Primary antibodies specific to anti-E-cadherin (1:200, Cell Signaling Technology), N-cadherin (1:400, Abcam), Smad2 (1:250, Abcam), and pSmad2 (1:1000, Abcam).

At indicated times, cells were fixed with pre-cooled 4% paraformaldehyde (Sigma) for 15 min, washed with PBS, and permeated with 0.5% Triton X-100 (Sigma) for 20 min at room temperature. After blocking with 5% bovine serum albumin (BSA, Sigma) for 30 min, cells were incubated with the following primary antibody overnight at 4°C: mIGF-1 (1:500 dilution, Cell Signaling Technology) and SIRT1(1:500 dilution, Cell Signaling Technology), and then with the diluted fluorophore-conjugated secondary antibody (Abcam, USA) for 1 h at room temperature. The cells were stained with DAPI (Abcam) according to the manufacturer's instruction. Finally, images were captured using a fluorescence microscope with a digital camera (MF52, Mshot) and analyzed with Image-Pro Plus version 6.0 software (Media Cybernetics, USA).

Cells in the dish were fixed with 4% paraformaldehyde and incubated with a primary antibody against p65 overnight at 4 °C. After washing several times with phosphate-buffered saline (PBS), the cells were incubated with an appropriate fluorophore-conjugated secondary antibody (Abcam) in the dark and counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, Shanghai, China). Images were captured using a confocal laser-scanning microscope (Leica TSC SP8, Wetzlar, Germany).