Lsm 510 meta nlo microscope

Mice were anesthetized with 3% isofluorane (2 L/min O₂) and transcardially perfused with 20 mL of ice-cold PBS, followed by 20 mL of ice-cold 4% PFA. Following fixation, brains were immediately removed and further fixed overnight in 4% PFA at 4 °C. Brains were processed in paraffin wax and sliced at 5 µM using a microtome. Following antigen retrieval, sections were washed in TBS + 0.1% triton X-100 and blocked for 2 h at RT in TBS, 0.1% triton X-100, 10% goat serum, and 5% BSA. Following three washes, slices were mounted in Vectorshield hardset mounting medium with/without DAPI (Santa Cruz). Antibody and cell count analysis details are shown in SI Appendix. All images were taken using a Zeiss LSM 510 META NLO microscope with ZEN 2009 software (Zeiss).

HT22 cells originating from the mouse hippocampus were purchased from ATCC (Manassas, VA, USA). They were cultured in Dulbecco’s modified Eagle’s medium as described previously [61 (link)]. CVE was dissolved in culture medium and HT22 cells were incubated with various concentrations (1–200 μg/mL) of CVE for 60 min. Thereafter, cells were harvested and assayed using the WST-1 assay kit (Sigma-Aldrich, St. Louis, MO, USA) to observe the conversion of tetrazolium salts into formazan by viable cells. Formazan fluorescence intensity was measured using a Fluoroskan ELISA plate reader (Labsystems Multiskan, Helsinki, Finland) as described previously [61 (link)]. Oxidative stress and DNA fragmentation induced by CVE was assessed 60 min after CVE treatment (50, 100, or 200 μg/mL). HT22 cells were incubated with 20 μM DCF diacetate (DCF-DA) to convert DCF-DA to DCF and cells were fixed for 3 h after 20 min of DCF-DA incubation. DNA fragmentation was visualized using a TUNEL staining kit (Sigma-Aldrich). DCF- and TUNEL-positive cells were observed by confocal fluorescence microscopy using an LSM 510 META NLO microscope (Carl Zeiss GmbH, Jena, Germany). DCF and TUNEL fluorescence intensities were measured using a Fluoroskan ELISA plate reader (Labsystems Multiskan).

Oxidative stress in HT22 cells was induced by exposure to 1 mM H₂O₂, and 50 μg/mL CVE was added to HT22 cells simultaneously with H₂O₂. Ten minutes after H₂O₂ and CVE treatment, HT22 cells were incubated with 20 μM DCF diacetate (DCF-DA) to convert DCF-DA to DCF. To measure ROS formation, cells were fixed for 3 h after H₂O₂ and CVE treatment. Cells were stained with a TUNEL staining kit (Sigma-Aldrich, St. Louis, MO, USA) to detect DNA fragmentation induced by H₂O₂. DCF- and TUNEL-positive cells were observed by confocal fluorescence microscopy using an LSM 510 META NLO microscope (Carl Zeiss GmbH, Jena, Germany). DCF and TUNEL fluorescence intensities were measured using a Fluoroskan ELISA plate reader (Labsystems Multiskan). Cell viability was measured using the WST-1 assay 5 h after H₂O₂ and CVE treatment, as described above. In addition, apoptosis and anti-apoptotic factors, including Bax and Bcl-2, were assayed by western blot. Briefly, cells were harvested 6 h after H₂O₂ treatment and lysed with ice-cold radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, MA, USA). Western blotting for Bax and Bcl-2 was performed as described previously [45 (link)]. Antibodies to Bax, Bcl-2, and β-actin were purchased from Abcam (Cambridge, UK) and were used at 1:2000 dilutions.