Thunder imager tissue

The goblet cells loss was determined by performing PAS staining analysis on colon tissues. The experiments were performed using a Periodic Acid-Schiff Staining Kit (C0142S, Beyotime, China) according to the manufacturer’s protocol. Finally, the images of sections (4 μm thick) were observed with an optical microscope (THUNDER Imager Tissue, Leica, Germany).

Livers were fixed for 24 h in 10% neutral buffered formalin, dehydrated, embedded in paraffin and cut into 3 µm-thick sections. Tissue sections were stained with hematoxylin &eosin & saffron (HES) with Leica autostainer for preliminary analysis. Tumor differentiation and characteristics were reviewed by an expert pathologist (BR). Slides were digitally processed using the Nanozoomer scanner (Hamamatsu).
Immunofluorescence assays were performed after deparaffinization and antigen retrieval in citrate buffer pH6.0 (Sigma Aldrich). The Click-iT AF488 (ThermoFisher Scientific) EdU detection reaction was performed according to the manufacturer’s instructions. Labeling of c-Myc-positive cells was performed with human c-Myc antibody (Abcam rabbit ab#32072 dilution 1:200) and anti-rabbit secondary antibody (Thermofisher anti-rabbit IgG AF633, dilution 1:1000). Images were acquired using the Thunder Imager Tissue (Leica) microscope. Quantitative image analysis was performed using the QuPath v0.4.4 software (Bankhead et al, 2017 ). Foci were hand drawn and positive cell detection were performed using nuclear staining as basis of cell recognition.

Tumors were harvested from the mice in different groups, fixed with 4% paraformaldehyde for at least 24 h, dehydrated through gradient sucrose solution, and frozen in optimal cutting temperature (OCT) medium. Tumors were cut via a cryotome, mounted on slides and stained with primary antibody overnight at 4℃. After washing three times in TBST, secondary antibodies were incubated for 1 h at room temperature. Images were acquired on fluorescence microscopy (THUNDER Imager Tissue, Leica).

Peritoneal macrophages and brain microglia were isolated and prepared from eGFP transgenic mice as previously described 46 (link). GL261 GBM cells were stained with Hoechst 33342. Then cancer cells (4×10⁵) were co-cultured with cultured peritoneal macrophages or brain microglia (1×10⁵) in serum-free medium containing NAcp@IgG or NAcp@CD47 and enzymes. After incubation for 30 min at 37℃, phagocytosis was studied by fluorescence microscopy (THUNDER Imager Tissue, Leica) and flow cytometry (FACSCantoTM II (Becton Dickinson).

Vglut2 cre x GCaMP6s animals were anesthetized with isoflurane and decapitated. Within 5 min of euthanasia, the L3/L4 spinal segments were removed and placed in OCT, flash frozen, and kept on dry ice until cryosectioning. Spinal cord was then sectioned in the transverse plane at 15 μm thickness, mounted on slides, and were probed for GCaMP6s and Vglut2 using RNAscope Multiplex Fluorescent Assay (Advanced Cell Diagnostics, 320851) using: Mm-Slc17a6 (Cat No. 319171) for Vglut2 and Mm-GCaMP6s-O1 (Cat No. 557091) for GCaMP6s. After hybridization and amplification slides were mounted with Prolong Gold with DAPI. Sections were then imaged using an upright epifluorescence microscope (Leica THUNDER Imager Tissue) using a 20x/0.80 NA air lens at 3883×3886 resolution (0.32 μm per pixel) and analyzed offline with FIJI. A positive cell required a clearly defined nucleus and fluorescent signal forming a ring around the nucleus, as shown before^{67 (link)}. 60-76 GCaMP6s+ nuclei were quantified within the superficial dorsal horn per mouse.

IHC staining was introduced to determine the ki-67 expression in colon tissues. The tissues (4 μm thick) were fixed using 4% paraformaldehyde fix solution (P0099; Beyotime, China) and embedded in paraffin, followed by cutting into 4 μm sections with a freezing microtome (FS800, Shenzhen Rayward Life Technology Co., Ltd, China). Ki-67 rabbit mAb (A11005) and biotin-labeled goat anti-rabbit IgG (H+L) were incubated with sections successively according to the procedure described in previous report [27 (link)]. Finally, the images of sections were observed with an optical microscope (THUNDER Imager Tissue, Leica, Germany).

The cells were fixed at 4% paraformaldehyde prepared in cytoskeletal buffer for 10 min at 37 °C [21 (link)] and washed with PBS 3 times. Then, the cells were blocked by bovine serum albumin for 1 h. Then, the primary anti-TRPC1 (1:1000; cat. no. #DF12783, Affinity Biosciences) and anti-TRPV4 (1:1000; cat. no. #DF8624, Affinity Biosciences) were incubated at 4 °C overnight, and the fluorescent secondary antibody Alexa Fluor 488-labeled Goat Anti-Rabbit IgG (H + L) (1:500; cat. no. A0423; Beyotime Biotechnolog) was used to incubate at room temperature and away from light for 1 h. Finally, the 4',6-diamidino-2-phenylindole (DAPI, Sigma, USA) was used for re-staining the nucleus for 5 min and observed under THUNDER Imager Tissue (Leica, Germany).