Axio observer z1 inverted phase contrast fluorescence microscope

COS-1 stable cells expressing GRTH were seeded on sterile coverslips kept inside a six-well cell culture plate at a concentration of 0.3 × 10⁶/well. The plate was kept overnight at 37 °C at 5% CO₂ to allow the cells to adhere to the coverslip. Peptides PEP1, PEP2, CP1, CP2, and CP3 at a concentration of 20 μM were added to each well. After 4 h of incubation with respective peptides, coverslips were carefully taken out from the cell culture plate and washed gently with 1× PBS. The cells were further fixed with 50 and 100% methanol for 1 min each and mounted inversely on a microscopic slide using ProLong Diamond Antifade Mountant with DAPI (Thermo Fisher Scientific). The slides were observed under the microscope and cells were microphotographed using Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence Microscope and Zen pro software.

Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence Microscope was used to capture fluorescence images. The microscope is equipped with a LED light source (Zeiss Colibri 7). The probe was visualized with a Cy7 filter (excitation: LP 710 nm, emission: LP 810 nm, beam splitter: 760nm) and fluorescence signals were captured with a digital camera (Hamamatsu, Model: C11440-42U30) and are shown with a pseudocolor of red. 10X objective lens and an additional 10X eyepiece were used with total magnification of 100X. Images were further processed with the ZEN 2.3 software (Blue version, Zeiss).

The redox-sensitive fluorescent dye chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetates (CM-H2DCFDA, ThermoFisher Scientific, #C6827) was used to measure intracellular ROS accumulation. UACC257 melanoma cells were cultured on a glass bottom dish and treated with the indicated siRNAs. Forty-eight h post siRNA treatment, 2 μM CM-H2DCFDA in PBS/5% FBS was added and the samples were incubated at 37°C for 30 min to assess overall ROS production. Subsequently, the cells were incubated with 5 μM MitoSOX Red (ThermoFisher Scientific, #M36008) in PBS/5% FBS at 37°C for 10 min, washed with HBSS, and analyzed by immunofluorescence imaging (Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence microscope). The results were normalized to cell numbers, which were determined by nuclear staining with 1 drop per ml of NucBlue (ThermoFisher Scientific, #R37605) at 37°C for 15 min.

For whole mount preparation, individual hair follicles were fixed with 4% paraformaldehyde (PFA) (Thermo Fisher Scientific, 50980487) for 30 minutes at room temperature and incubated for 1 hour in a blocking buffer containing 10% goat serum (Sigma Aldrich, G9D23) and 5% BSA (Sigma Aldrich, A3294). The hair follicles were subsequently incubated with diluted primary antibodies overnight at 4°C. TRP-2 (abcam, ab74073; 1:100), Ki67 (abcam, ab15580; 1:100), TRP-1 (abcam, ab186929; 1:100), and γH2AX (abcam, ab81299; 1:100) were used as the diluted primary antibodies. The hair follicles were then incubated with 1:500 diluted secondary antibodies for 1 hour at room temperature. Alexa Fluor 594 (goat anti-rabbit IgG; Thermo Fisher Scientific, A-11012) and Alexa Fluor 488 (goat anti-rabbit IgG; Thermos Fisher Scientific, A-11008) were used as the diluted secondary antibodies. Nuclei was labeled with VECTASHIELD Mounting Medium containing DAPI (Vector Laboratories, H-1200).
In the TUNEL assay, cell death was detected by TUNEL staining (TdT-mediated dUTP-digoxigenin nick end labeling technique) using the “in situ cell death detection kit” (Roche Diagnostics, 11684795910). Images were captured using confocal microscopy (Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence microscope). Standard microscopy techniques were used to adjust brightness, contrast, focus, and image capture.

Quantitative image analysis was conducted to assess morphological changes of TP53^KO and TP53^R249S liver organoids. Cells seeded in black 96-well culture dish were maintained in culture medium supplemented with 40 μM Calcein AM (Invitrogen) and 2 μg/mL of Hoechst 33342 (Invitrogen) for 2 hours at 37 °C. Fluorescent images were captured by the Cellomics ArrayScan VTI (ThermoFisher Scientific) under standard culture conditions (37°C and 5% CO₂). Morphology counting was conducted by two individuals based on 20 fields of each well, and only organoids larger than 50 μm were considered.
For time-lapse live-cell imaging, organoids were plated in 35mm culture dish. On day 4, the dish was placed into the culture chamber of the Zeiss Axio Observer Z1 Inverted Phase Contrast Fluorescence Microscope. Images were taken at a time interval of 10 min for 24 hours. The images were later processed and assembled into videos using the Zen software.