Dp70 camera

For the investigation of nucleus morphology and chromatin condensation, DAPI staining was carried out. Both cell lines were cultivated in the 8-well chamber slides (5 × 10³ cells/well) and incubated for 24 h. Afterward, cells were treated with synthesized formulations and SUN. Subsequently, 24 h post-treating, cells were washed, fixed, and permeabilized using 0.1% (W/V) Triton X-100, stained with 200 μL/well of DAPI (200 ng/mL), and finally, PBS was added to the wells to keep the cells hydrated. The cells were investigated with Olympus IX81 inverted fluorescence microscope and an Olympus DP70 camera (Olympus Corporation, Tokyo, Japan).

Dissected tissues and cell cultures were fixed in 4% paraformaldehyde. Tissue sections (5 μm thick) were deparaffinized and rehydrated as previously described in ref. ^{60 (link)}. For hematoxylin and eosin staining, sections were immersed in hematoxylin (Merck) and eosin (Merck) for 4 and 2 min, respectively. Evaluation of lipid content was performed using Oil red O staining. Quantification of lipid content was performed after isopropanol solubilization at 510 nm. The histological study of the stained sections was carried out using a Leica DM6000B microscope equipped with a DFC390 camera (Leica, Barcelona, Spain) and an Olympus IX71 with an Olympus DP70 camera (Olympus). Adipocyte size was quantified with ImageJ software (National Institutes of Health).

Five μm sections of formalin-fixed, paraffin-embedded murine lung tissue were stained with hematoxylin and eosin (H&E) and Periodic acid-Schiff (PAS) (AML Laboratories, Inc. Baltimore, MD) and photographed using Olympus-DP70 camera on Olympus-BX41 microscope. Histopathologic evaluation of H&E stained lung sections from at least 4 mice per group was performed by a pathologist in a blinded fashion using a semi-quantitative scoring system on a Nikon Eclipse microscope. Peribronchiolar and perivascular inflammatory cellular infiltration were scored separately as follows: 0, no or occasional cells; 1, few or loosely arranged cells; 2, focal involvement of lung parenchyma with majority of airways or vessels having rings (partial or complete) of inflammatory cells one cell layer deep; 3, patchy involvement of lung parenchyma with majority of airways or vessels having rings (partial or complete) of inflammatory cells two to four cell layers deep; 4, extensive involvement of lung parenchyma with majority of airways or vessels having rings (partial or complete) of inflammatory cells more than four cell layers deep. Individual scores for peribronchiolar and perivascular inflammation were added together for a total maximum score of 8.

Specimens were decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 1 month, dehydrated and embedded in paraffin. Sections (5 μm) were cut and stained with haematoxylin and eosin (H&E) or TRAP by Wuhan Servicebio Technology Co., Ltd. For immunohistochemistry analysis, sections were blocked in 3% BSA for 30 min and then incubated with primary antibodies against TAK1 (ab109526, Abcam, USA) and OCN (23418-1-AP, ProteinTech, Wuhan, China) at a 1:100 dilution. Antigen retrieval was performed in 95 °C citrate buffer (pH 6) for 10 min, and then sections were incubated with primary antibody at 4 °C overnight. After processing with an ABC detection kit (Vector Laboratories, Burlingame, CA), sections were visualized under an Olympus BX51 light microscope equipped with an Olympus DP70 camera (Olympus Co., Tokyo, Japan) and quantified using ImageJ software version 1.53 (US National Institutes of Health).

The DAPI staining assay was conducted to detect possible occurrence of nucleus condensation in the treated cells. Briefly, the treated cells were fixed with the freshly prepared ice-cold paraformaldehyde (4%) and then exposed to 0.1% Triton X-100 in PBS for 5 min for permeabilization. They were subsequently stained with DAPI (1 μg/mL in PBS) for 5 min in the dark. After removing the surplus stain, the cells were washed (3×) using 0.1% Triton X-100 in PBS. The image acquisition was performed by Olympus IX81 invert fluorescence microscope equipped with Olympus DP70 camera (Olympus Corp., Tokyo, Japan) as described previously [27 (link)].

Livers, lungs and kidneys were fixed in 10% formalin and embedded in paraffin. Thin sections stained with hematoxylin and eosin were evaluated in a blinded fashion by a pathologist (BSP). Sections from control slides were first examined to determine baseline (score of 0). Then, the rest of the slides were screened in a blinded fashion and given a score of either 1 or 2 based on the extent of inflammatory changes. For each group, 6 to 16 individual sections from different mice were examined and scored. An Olympus AX70 research microscope fitted with an Olympus DP70 camera (Olympus America, Center Valley, PA, USA) was used to acquire images.