Axio observer z1 microscope

Tumor tissues from control and PPLGM-treated mice were collected and fixed for 24 hours in formaldehyde. Paraffin-embedded 5 μm thick sections of tumor tissues were prepared. Sections were deparaffinized with histoclear and ethanol, followed by antigen retrieval in 10 mM sodium citrate buffer (0.05% Tween 20, pH 6.0) using an autoclave method. The sections were blocked for 20 minutes in blocking buffer (10% normal goat serum in TBST) and incubated with primary antibodies (Ki-67 [1:100] and 8-OHdG [1:20]) overnight at 4°C. The next day, sections were incubated with CF633-conjugated goat anti-mouse secondary antibody (1:250) for an hour at room temperature, and were visualized using a Zeiss inverted Axio Observer Z1 microscope after mounting a coverslip using Hardset Mounting media with DAPI (Vector Labs; Burlingame, CA).

BAX translocation was quantified in MEFs and NRCMs by the presence of mitochondrial-localized BAX puncta as previously described^{52 (link)}. Cells were treated with 2 μM STS for 3 h (MEFs) or 5 h (NRCMs). For MEFs, mitochondria were identified using MitoTracker Red (Invitrogen, M22425) before fixation according to the manufacturer’s protocol. Cells were fixed in 4% paraformaldehyde, permeabilized in PBS with 0.5% Triton X-100 and blocked with PBS with 2% BSA and 1% Triton X-100. Immunostaining was performed using BAX antibody and Alexa Fluor 488 secondary antibody. For NRCMs, mitochondria were identified by co-immunostaining with ATP5α antibody and Alexa Fluor 546 secondary antibody. After counterstaining of nuclei with either Hoechst 33342 or DAPI (Vector Laboratories, H-1500) slides were visualized using Axio Observer.Z1 microscope (Carl Zeiss) at ×200 magnification. Five fields per treatment (~250–350 cells per field) were randomly selected and the percentage of cells with mitochondrial BAX puncta was scored.