Fluorescence microscope system

In a subset of extracellular recording experiments, the tip of the micropipette was coated with DiI (DiIC18(3), life technologies) for placement reconstruction (Figure 1A). At the end of the experiment, animals were deeply anesthetized with pentobarbital sodium and perfused transcardially with PBS followed by 2.5% paraformaldehyde. Brains were post-fixed and cryo-preserved in 2.5% paraformaldehyde and 30% sucrose in PBS, respectively. Subsequently, coronal sections of the brain were obtained (30 μm), mounted on microscope slides and covered with cover glass. Visualization was carried out under a fluorescence microscope system (Leica).

At the end of the antitumor study, the mice were sacrificed and major organs, as well as tumors were collected. The tumor tissues were frozen for cell apoptosis, collagen, and CD31 staining. For collagen staining, sections were fixed, blocked, and incubated with rabbit anti-mouse collagen antibody (1:50) at 4 °C overnight followed by incubating with Alexa Fluor 647-conjugated secondary antibody (1:100) for 1 h at room temperature. For TUNEL analysis, experiments were carried out according to manufacturer’s standard protocol. For the immunohistochemistry of CD31, sections were fixed, blocked, and incubated with rabbit anti-mouse CD31 antibody (1:50) at 4 °C overnight followed by incubating with the FITC-conjugated secondary antibody (1:100) for 1 h at room temperature. Cell nuclei were stained with DAPI and the sections were covered with a coverslip. The sections were scanned by Leica fluorescence microscope system. All the images were captured at ×200 magnification. The major organs, including heart, liver, lung, and kidney were fixed in 10% formalin for H&E staining.

For the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, cells were fixed in 4% paraformaldehyde, incubated with permeabilization buffer containing Triton X-100 (Sigma), fluorescence-labeled reagent (45 μl) and terminal deoxynucleotidyl transferase (5 μl) (cat. #, 24529300; Sigma) for 1 h at 37°C. The nuclei were labeled with 4′,6-diamidino-2-phenylindole (Solarbio) for visualization. A fluorescence microscope system (Leica, Germany) was used to observe and capture images of the cells.