Eclipse e100

ATP content was determined using the Enhanced ATP Assay Kit. Lysed cells or tissues were centrifuged at 12,000×g for 10 min, and the supernatant was collected. FLS were lysed with 200 μL of ATP-lysate reagent and centrifuged at 12,000×g for 10 min. Subsequently, 20 μL of the supernatant was transferred into a 96-well black lightproof plate, and then 100 μL of ATP detection reagent was added to each well for 5 min. The chemiluminescence of each well was monitored on a microplate spectrophotometer (Envision, PerkinElmer, Waltham, MA) in luminance mode within 30 min.
The ΔΨm was measured using Rhodamine 123. FLS were rinsed with PBS twice, and 2 mL of Rhodamine 123 solution (5 μm) was added to each well and dyed at 37 °C for 20 min in the dark. The staining solution was discarded, and the FLS were washed 3 times with D-Hanks. The change in ΔΨm was observed under a Nikon Eclipse E100 (Nikon, Japan), and the fluorescence intensity reflected the level of ΔΨm.
ROS levels were detected using 2′,7′-dichlorofluorescein diacetate according to the manufacturer's protocol. ROS working solution (10 μm) was added to FLS and incubated at 37 °C for 15 min in the dark. Then, the cells were washed with D-Hanks twice and photographed under a Nikon Eclipse E100 (Nikon, Japan). The average fluorescence intensity of the cells was analyzed using Image-Pro Plus software (Media Cybernetics Inc.).

Renal fibrosis was assessed via Masson staining. Dewaxed slices were soaked in Masson dye solution (Servicebio, G1006, Wuhan, China) according to the manufacturer's instructions and then observed under a microscope (Nikon Eclipse E100, Nikon, Japan), and images were acquired and analyzed (Nikon DS-U3, Nikon, Japan). Collagen fibers were stained blue, while muscle fibers, cellulose, and red blood cells were stained red. The fibrotic area (%) identified via Masson staining was analyzed using ImageJ.
The morphology of the spleen and kidneys was observed via HE staining. Dewaxed slices were stained with an HE dye solution set obtained from Servicebio (Servicebio, G1003, Wuhan, China) following the standard steps. The stained spleens and kidneys were observed under a microscope (Nikon Eclipse E100, NIKON, Japan), and images were acquired and analyzed (Nikon DS-U3, NIKON, Japan). The nuclei were stained blue, and the cytoplasm was stained red.

The young spikes of WT and mutant ptsd1 were fixed in FAA solution (5 mL of formalin, 5 mL of acetic acid and 90 mL of 70% ethyl alcohol). The samples were dehydrated, embedded in paraffin and sectioned with a rotary microtome as described by Geng et al. [70 (link)]. The cell morphology was observed after the tissues were stained with saffron and solid green (G1031, Servicebio, Wuhan, China). The distributions of starch and protein were observed after the tissues were stained with Periodic Acid-Schiff and Naphthol Yellow S (G1068, Servicebio, Wuhan, China). Photos were taken with a camera (Nikon Eclipse E100, Tokyo, Japan) and analyzed with CaseViewer software.

Brain tissues were embedded in paraffin and sectioned. Then sections were processed as follows: Xylene for 20 min, two times; 100% ethanol for 5 min, two times; 75% ethanol for 5 min; and tap water rinsing. Sections were subjected to hematoxylin–eosin (HE) and toluidine blue (Nissl) staining. Hematoxylin–Eosin Staining Kit (G1003; Wuhan Servicebio Technology Co., Ltd., Wuhan, China) was used for HE staining, and Toluidine blue staining solution (G1032; Wuhan Servicebio Technology Co., Ltd.) was used for Nissl staining. Sections were finally sealed with neutral gum and examined under an upright optical microscope (NIKON ECLIPSE E100) for image acquisition.

After removal, scapular BAT (about 3 x 3 x 3 mm³) of sixteen animals (n = 4 per group) was immediately washed with PBS solution twice and stored in paraformaldehyde. 24 h later, the BAT was washed, dehydrated in graded alcohol concentrations, cleared in xylol and embedded in paraffin. The paraffin blocks were cut serially to provide 5 μm-thick sections. Serial sectioning was performed until reaching the maximum number of sections possible. Sections representative of each specimen were selected and stained with hematoxylin-eosin dyestuff. The stained slice was dehydrated with pure alcohol and made transparent using xylene. The transparent slice was then placed onto a slide and sealed with a coverslip. Images were captured using a microscope (ECLIPSE E100, NIKON, Japan) coupled to a video camera (K-Viewer 1.5.5.10 x64, KFBIO, China). Ten slices were randomly selected from each animal for the histometric analysis, and one image representative of its respective group was selected to present in this paper.