Fluoview fv1000 confocal microscope system

3T3-L1 cells grown on a cover slip were fixed with 3% (w/v) paraformaldehyde for 10 minutes at room temperature or with 100% methanol for 2 minutes at -20°C, and immunostained with anti-β-tubulin and anti-GEF-H1 primary antibodies and Alexa Fluor 488- or Alexa Fluor 568-conjugated secondary antibodies as described previously [13 (link)]. The actin filaments were stained with Alexa Fluor 568-conjugated phalloidin (Thermo Fisher Scientific). Cells were also stained with 4',6-diamidino-2-phenylindole (DAPI) to visualize the nuclei. Immunofluorescence images were obtained with FluoView FV1000 confocal microscope system (Olympus, Tokyo).

The TUNEL assay to detect DNA fragmentation was performed according to the manufacturer’s instructions to determine the percentage of apoptotic cortical neurons. This assay is based on the nicks in the DNA specifically identified by TdT. This enzyme catalyzes the addition of dUTPs labeled with a fluorescent marker. The normal C57, wild-type and TRPC5 KO mice cerebral slices were fixed in a 4% paraformaldehyde solution for 25 min at 25 °C. After being washed three times, the neurons in the slices were permeabilized by a solution of 0.1% Triton X-100 and 0.1% sodium citrate for 5 min at room temperature. After equilibrating for 30 min in equilibration buffer at room temperature, the slices were treated with BrightRed Labeling Mix (TdT and 12-dUTP) for 1 h at 37 °C in a humidified and dark chamber. After the reaction, neuronal nuclei were stained with 4’,6-diamidino-2-phenylindole (DAPI). Finally, apoptotic (red; at a wavelength of 620 nm) and total neurons (blue; at a wavelength of 460 nm) cells were counted using a FluoView FV1000 confocal microscope system (Olympus, Tokyo, Japan). The percentage of apoptotic neurons was calculated as follows: (apoptotic cell number/total cell number) × 100.

Annexin V is a Ca²⁺-dependent phospholipid-binding protein that has a high affinity for PS. It is widely used to identify cells undergoing apoptosis with exposed PS. FITC, a fluorochrome conjugated to annexin V, serves as a sensitive probe for flow cytometric analysis. An annexin V-FITC apoptosis detection kit was used to detect PS externalization. Briefly, HEK293 cells were pretreated with NPSS (control), hypotonic solution, daidzein (100 μmol/L), or LaCl₃ (100 μmol/L) for 15 min at 37 °C in an incubator. The cells were then washed twice with cold NPSS and incubated with 300 μL of binding buffer (0.01 M HEPES/NaOH at pH 7.4, 0.14 M NaCl, 2.5 mM CaCl₂). Annexin V-FITC (5 μL) was added to the cells and gently mixed and incubated for 15 min at room temperature in the dark. Finally, another 200 μL of binding buffer was added to the cells. The resultant images were visualized using a FluoView FV1000 confocal microscope system (Olympus, Tokyo, Japan).