Ix73 microscope

Treg cells were fixed in 4% paraformaldehyde for 5 minutes, washed with PBS twice, and ruptured in Triton-X-100 at room temperature for 30 min. The cells were incubated for 30 min with immunofluorescent blocking solution. Next, they were incubated at 4°C overnight with different primary antibodies. After washing the primary antibodies, the cells were incubated with different secondary antibodies at room temperature for 1 hour. Then the cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and washed with PBS three times; the cells were added to antifluorescence quencher and covered with a cover slip, and the protein expression was detected by Olympus IX73 microscope (Tokyo, Japan). Image J was used for the quantitative analysis. The above antibodies were as follows: primary antibodies: Rabbit anti-mouse β-actin, Rabbit anti-mouse PI3K P110α (all 1 : 100, CST, USA), Rabbit anti-mouse C-JUN antibody, Rabbit anti-mouse AKT, and Rabbit anti-mouse PI3K P85α (all 1 : 100, Abcam, USA); secondary fluorescently labeled antibodies: Donkey anti-rabbit antibody (Alexa Fluor 488) and Donkey anti-rabbit antibody (Cy3) (all 1 : 200, Jackson, USA). Testing equipment was Olympus IX73 microscope (Tokyo, Japan).

The paraffin-embedded gastric tissues were dewaxed, rinsed with phosphate-buffered saline (PBS, pH 7.4) three times for 5 min and treated with 10% FBS at 37 °C for 30 min. Sequentially, the gastric tissues were dewaxed with dimethyl benzene and an ethanol gradient, washed with phosphate-buffered saline (PBS) for 3 min each time for a total of three times and treated with 10% FBS at 37 °C for 30 min. Next, sections were incubated with primary antibodies against Keap1 (1:500, ab196346, Abcam, Cambridge, UK), Nrf2 (1:200, ab137550, Abcam, Cambridge, UK), TLR4 (1:500, ab22048; Abcam, Cambridge, UK) and NF-κB (1:500, ab16502; Abcam, Cambridge, UK) in a humidified chamber at 4 °C overnight. The sections were incubated with HRP-labelled secondary antibodies for 30 min after being washed with PBS three times. Then, 3,3′-diaminobenzidine (DAB, Sigma-Aldrich, St. Louis, MO, USA) was used as a substrate to visualize positive staining. Finally, images were obtained by using an IX73 Olympus microscope equipped with a DP80 camera (Olympus Corp., Tokyo, Japan).

Oil Red O (ORO) staining was used to highlight the amount of neutral lipids accumulated during adipogenic differentiation. Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich, Darmstadt, DE) at 4 °C, permeabilized with 2% BSA solution with 0.1% Triton X100 for 1 h and stained with 5 mg/mL Oil Red O solution for 2 h at room temperature. The lipid droplets of the cells were observed at 7 and 21 days after the induction of adipogenesis.
Alizarin Red S (ARS) staining was performed to highlight the degree of mineralization of ECM during in vitro osteogenesis. For the ARS staining, the cells on the materials were fixed in 4% paraformaldehyde (Sigma-Aldrich, Darmstadt, DE), washed with PBS and stained with 2% ARS staining solution. The mineral deposits stained in bright red were observed under the microscope on days 14 and 21 after the induction of osteogenesis.
The images were acquired by IX-73 Olympus microscope. The quantification of ARS and ORO stained elements was made with Image J software and plots were obtained with GraphPad Prism version 6 for Windows.

A Live/Dead^® viability kit (L3224, Invitrogen™, Gloucester, UK) was used to assess cell viability, as per manufacturer’s protocol. Briefly, at each timepoint, cells were washed with HBSS and incubated with a solution containing 2 µM calcein-AM and 4 µM ethidium homodimer in HBSS for 30 min at 37 °C in a 5% CO₂ humidified atmosphere. DMSO was added for positive dead cell controls. Excess dye was removed by washing with HBSS. Samples were visualised using an inverted IX73 Olympus microscope (Olympus Corporation, Tokyo, Japan). At least 5 regions of interest (ROI) per image were selected. Alive and dead cells were counted with ImageJ software (NIH, Bethesda, MD, USA) and percentage cell viability was calculated as alive cell number against total cell number.

Primary cultures of microglia were obtained from the CX3CR1-eGFP transgenic mice. The same concentrations of inhibitors were used in both the inserts and in the 24 well culture plate (#353047, Corning, New York, NY, USA) to assess the rate of migration. The cultures were incubated for 3 h, at 37 °C, 5% CO₂, allowing the cells to migrate through the 8 µm pores. Afterwards, the inserts were fixed with 4% paraformaldehyde (PFA) (#252549; Sigma, St. Louis, MO, USA) for 5 min, stained with 2 µg/mL of Hoechst (#33342, Sigma, St. Louis, MO, USA) in PBS for 20 min in the dark and pictures were taken using an IX73 Olympus microscope and CellSens software. The images were processed with the Fiji software, all the experiments were replicated from at least 3 different animals in sham or SNI conditions. The migration rate was analyzed by counting the Hoechst-stained nuclei from the CX3CR1-eGFP microglial cells that migrated through the insert and the data are represented as normalized values. The n values represent the number of pictures analyzed for each condition.