Ckx41 inverted microscope

ELISA and tissue section were employed to investigate the inflammation and liver damage of TAA-treated mice. The ELISA kit used for analysing plasma levels of proinflammatory cytokine tumour necrosis factor-α (TNF-α), high mobility group protein B1 (HMGB-1), and interleukin-6 (IL-6) levels was purchased from BioLegend (San Diego, CA, USA). An antibody used for the immunohistochemical (IHC) detection of TNF-α expression in mouse liver was obtained from R&D Systems (Minneapolis, MN, USA). Rat anti mouse IL-6 antibody was purchased from BioLegend. (San Diego, CA). Goat anti mouse HMGB1 antibody was purchased from Chondrex. (Redmond, WA). DNA staining dye 4′,6-Diamidine-2′-phenylindole dihydrochloride (DAPI) was purchased from Sigma-Aldrich. Tissue section, haematoxylin and eosin staining (H&E staining), and immunohistochemistry (IHC) were performed following previously described methods^{27 (link),40 (link)} using an IHC kit purchased from Thermo Fisher Scientific (Fremont, CA, USA). Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan) was used to capture images. The specific staining signals (e.g. green channel of TNF staining) were quantified using ImageJ software (NIH, Bethesda, MD, USA).

For the β‐galactosidase staining, the cells were seeded on 24‐well plates at 20 000–30 000 cells per well 24 h prior to treatment. The cells were then treated with test compounds for 72 h, fixed, and stained with Senescence β‐galactosidase Staining kit (#9860, Cell Signaling Technology, Danvers, MA, USA) according to the manufacturer's instructions. Briefly, the cells were rinsed once with PBS, fixed with solution provided in the kit for 15 min at RT, and rinsed again two times with PBS. Staining solution was then added to the cells, and the well plate was sealed and incubated for overnight at 37 °C in normal atmosphere. The cells were examined and imaged with Canon EOS 600D camera (Canon, Tokyo, Japan) on Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan) on the following day.
β‐Galactosidase activity was measured utilizing 96‐well Cellular Senescence Assay kit (CBA‐231; Cell Biolabs, San Diego, CA, USA). For this assay, cells were seeded on a 96‐well plate at 2000 cells per well, let to attach overnight, and treated with the compounds for 72 h. After treatment, the cells were rinsed with PBS and lysed. Lysates were centrifuged and the supernatants collected and incubated with fluorescent substrate for 2 h at 37 °C, protected from light. The reaction was then stopped and the fluorescence (Ex 360 nm/Em 465 nm) measured with Victor² microplate reader (PerkinElmer).

Colony-forming efficiency of ESC_endo and ESC_cyst was assessed using CFU-F in which cells were seeded in six-well plates at optimized densities of 200 cells/well in complete DMEM-LG growth medium. The growth medium was changed every 4 days. Following 21-day culture in 37°C with 5% CO₂, the growth medium was removed and the cells were washed twice with PBS. The cells were then fixed and permeabilized with 100% cold methanol for 5 minutes at RT. After washing the cells twice with PBS, they were stained with 1% Eosin in PBS for 20 minutes at RT, then rinsed twice with milliQ water, and visualized at 4x magnification under an Olympus CKX41 inverted microscope (Olympus, Tokyo, Japan). Colonies of cell aggregates of ≥50 cells were scored in the whole wells.

The cells were exposed to different treatments as per the experimental group. The medium and DMSO was added in the control group. As for model group, the cells were treated with optimized concentrations of MPP⁺ for 24 h. Then, the medium was added for 48 h. For the administration group, after incubation for 24 h at 37 °C, the cells were treated with the optimized concentrations of MPP⁺ for 24 h. Then, the cells were treated with the optimized concentration of XA for 48 h. The morphology of the cells in each group was observed under an inverted microscope (CKX41 OLYMPUS Inverted Microscope, Olympus Corporation, Tokyo, Japan), and the cells were photographed.

Haematoxylin and eosin were used to analyse graft-localised cell infiltration and inflammation. All images were acquired using a CKX41 Olympus inverted microscope (Tokyo, Japan) equipped with a DFK 31AU03 camera (The Imaging Source Europe GmbH, Germany) and IC Capture software (The Imaging Source Europe GmbH, Germany).

Anticancer activities of AuNPs against A549, CaCo-2, and MCF-7 cell lines were investigated using MTS assay protocol as described in the safety assay method. The morphological changes occurred in cancer cells, post AuNPs treatment, were inspected using CKX41 Olympus Inverted Microscope, Japan.