Virtual slide microscope vs120 s6 w

For immunofluorescence, the kidney tissues were fixed in 10% formaldehyde solution at 4 °C for 24 h, and were respectively dehydrated with 10, 20, 30% sucrose, followed by embedding in optimal cutting temperature compound (OCT). Then the 4 μm slices of kidney samples were incubated with primary antibody (F4/80, Santa, sc-377,009, USA, 1:200; Mincle, Santa, sc-390,806, USA, 1:200) at 4 °C overnight. After washing 3 times with PBS for 5 min, samples were incubated with Alexa Fluor® 488 (Cell Signaling Technology, #8878, USA; 1:200) and Alexa Fluor® 647 (Cell Signaling Technology, #8940, USA; 1:200) conjugated secondary antibodies at room temperature for one hour the next day. Finally, take the images with Virtual Slide Microscope (VS120-S6-W, Olympus, Japan).

Sections were incubated overnight at 4 °C with primary antibody: type II of collagen (Col II, 1:1000), Aggrecan (1:800), autophagy marker LC 3B (1:200), and Beclin1 (1:200) dilutions, respectively, prior to incubation with secondary antibodies, as described in the manufacturer’s instructions (MAIXIN.BIO, Fuzhou, China). Diaminobenzidine was then used to visualize the immunohistochemical reaction followed by being counterstained with hematoxylin. Images were scanned using the Virtual Slide Microscope (VS120-S6-W, Olympus, Tokyo, Japan). Dark brown cells or area were considered to be positive. The positive chondrocytes were counted semi-automated using Image-Pro Plus 6.0 Software, and area was measured using ImageJ Software, followed by analysis with GraphPad Prism version 5 [15 (link), 17 (link), 29 , 30 (link)].

To count the number of c-fos⁺, Nts⁺, Penk⁺, Crhr²⁺ and CTB⁺ cells, we collected 40 μm coronal sections of target brain regions for each mouse. The images were acquired with slide scanner (Olympus Virtual Slide Microscope, VS120-S6-W) and then cell counting was performed with custom-written MATLAB software.

For immunohistochemistry, TNF-α, IL-1β, IL-6, and F4/80 were tested in kidney samples on slices. First, the slices were dewaxed in xylene and rehydrated in gradient alcohol before antigen repair (citrate buffer pH 6.0, 95 °C for 10 min). Next, immunohistochemical staining of the tissue was performed according to the kit instructions. Briefly, the slices were incubated at 4 °C overnight with corresponding primary antibody (TNF-α, Santa, sc-52,746, USA, 1:200; IL-1β, Santa, sc-12,742, USA, 1:200; IL-6, Santa, sc-532,296, USA, 1:200; F4/80, Santa, sc-377,009, USA, 1:200) in 1% bovine serum albumin (BSA). The next day, after washing three times with PBS, the slices were treated with Biotin-Streptavidin HRP-based SPlink Detection Kits (ZSGB-Bio, PV-6000, China) and stained the nucleus with hematoxylin. Ultimately, all the slices were photographed with Virtual Slide Microscope (VS120-S6-W, Olympus, Japan).

To visualize the downstream projection brain regions of NAc neurons receiving BLA and PVT input, 50-μm brain sections were photographed with Olympus Virtual Slide Microscope (VS120-S6-W) using ×10 objective. We found that the major downstream projection brain regions are the VP, LH, and VTA. Three representative images were selected for each downstream brain region for every mouse, and the fluorescence intensity was calculated by ImageJ software. To quantify the co-localization of virus-expressing neurons with endogenous mRNA (D1/ D2) or CTB-647/CTB-488, three or four representative ×20 images of each mouse were selected. Co-localization was identified by eye.