Lsm 700 confocal microscope system

Cells transfected with mRFP-GFP-LC3 plasmid were seeded in 35 mm glass-bottom dishes and cultured overnight, followed by treatment with apatinib or control vehicle. After the indicated times, the cells were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min at room temperature. Then, the cells were washed with PBS and left in the dark before fluorescence microscopy analysis. Confocal images of cells were sequentially acquired with Zeiss AIM software on a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany). The autophagosomes are shown as yellow dots (RFP⁺GFP⁺), whereas the autolysosomes are shown as red dots (RFP⁺GFP⁻).

Endogenous peroxidase activity was quenched by incubation in 3% hydrogen peroxide in methanol for 30 min. The sections were blocked for 1 h with blocking solution (0.3% Triton X-100 and 5% bovine serum albumin in PBS). These sections were incubated overnight with primary antibody against TH (1:2000) and GFAP (1:1000). After washing in PBS, the sections were incubated in horseradish peroxidase-labeled goat anti-mouse/rabbit IgG antibody (1:1000) for 1 h at room temperature. The peroxidase reaction was performed in PBS using 3,3-diaminobenzidine tetrahydrochloride (DAB). Specimens were observed and counted using MicroBrightField Stereo Investigator software (Micro Bright Field, Williston, VT, USA) to measure the TH-positive cells and GFAP-positive cells.
For immunofluorescence, sections and cells were incubated with primary antibody against GFAP (1:1000) or GLT-1 (1:1000) overnight at 4 °C, and then labeled with an anti-rabbit secondary antibody conjugated with Cy3 or FITC (1:1000) in the dark for 1 h at room temperature. Cell nuclei were counterstained with 40, 60-diamidino-2-phenylindole (DAPI). Images were acquired by fluorescent microscopy (IX 70, Olympus, Japan). The cell confocal images were acquired with a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany).

Cy3-labeled specific probe to circSHKBP1 and FAM-labeled specific probe to miR-582-3p were designed and synthesized by RiboBio and the signals was detected by the FISH Kit (RiboBio) according to the manufacturer’s instructions. Cells were grown to the exponential phase and were 40–50% confluent at the time of fixation. After permeabilization (1 × PBS/0.5% Triton X-100), the cells were hybridized in hybridization buffer with specific probes to circSHKBP1, U6 and 18S at 37 °C overnight. The hybridization buffer was then gradually washed off with 4× SSC (including 0.1% Tween-20), 2× SSC and 1× SSC at 42 °C. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (RiboBio). Confocal images were captured using Zeiss AIM software and a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany).

The clonal analysis was performed as described previously (Singh et al., 2015 (link)). Briefly, for sparsely labeling the progenitor cells, 3-week-old mice carrying Gli1^creERT2; Rosa26^Tom with wild-type or Kcna1^-/- and Ntrk2^flox/flox or Ntrk2^flox/+ were injected with a single dose of 0.5 mg/kg of tamoxifen (Sigma, USA) dissolved in corn oil (Sigma, USA). At 8 weeks of age, mice were killed by an intraperitoneal injection of 150 mg/kg Zoletil + 20 mg/kg xylazine. Both hippocampi were dissected out and fixed overnight in a PBS solution containing 4% PFA. The hippocampi were rendered transparent in scale 0 for 2 days at 37°C, and then transferred to scale 4 for 1 day at 4°C (Hama et al., 2015 (link)). The whole-mount hippocampus was imaged in 3D using a Zeiss LSM700 confocal microscope system (Zeiss, Germany). Imaris9.7 (Oxford Instruments, Switzerland) was used to identify TdTomato-positive neural progenitor cells. The clonal clusters were defined as the TdTomato-positive cells within 100-μm radius of the clone center, as illustrated in Figure 7—figure supplement 1 (Singh et al., 2015 (link)).

Immunofluorescence staining was performed on the sectioned carotid arteries to assess the infiltration of inflammatory cells. Vascular tissue sections were blocked with 5% goat serum for 30 min at room temperature, then incubated with primary antibody CD68 (1:500; Abcam, Cambridge, England) overnight at 4°C. The secondary antibody used was Alexa Fluor 488 donkey anti-mouse (1:1000; Invitrogen, Life Technologies, Carlsbad, CA, USA). Sections were incubated with the secondary antibody in the dark for 2h at room temperature. The nuclei were then stained with DAPI. The localization of CD68, a monocyte/macrophage marker was assessed by fluorescence microscopy (ZEISS). Confocal images of sections were sequentially acquired with Zeiss AIM software on a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany). For quantification, three randomly selected high power fields (HPFs) (400× for IHC and immunofluorescent studies) were analyzed in each section. The mean number of positively stained cells per HPF for each rat was then determined by summation of all numbers.

The cy3-labeled circ_0072088 probe was purchased from GenePharma. A FISH Kit was purchased (Ribobio, Guangzhou, China) and performed accordingly. Afterward, Zeiss AIM software along with a Zeiss LSM 700 confocal microscope system (Carl Zeiss Jena, Oberkochen, Germany) was utilized to capture confocal images of cells.