Spinning disc microscope

To assess protein amounts in different subcellular domains, images used for comparison within an experiment were obtained with identical settings on a Zeiss Spinning Disc microscope and then used for quantification without any manipulation. A mask was constructed by manually outlining the cells in the image taken in the Rab5 or Lyso channel, for example. This mask was then applied to the image taken in the channel for the protein of interest, and the fluorescence was measured. Local background correction was performed by moving the mask to measure fluorescence at a representative nearby region and then subtracting this value from that of interest. All bars represent mean ± s.d. from 20 individual cells. Statistical analysis was performed using GraphPad Prism software.
To assess protein amounts in different wing imaginal discs, all wing discs from third instar larvae were dissected, fixed, stained in the presence or absence of detergent, and imaged in parallel using the same confocal microscope settings. To quantify Ihog, Ptc, and GFP-staining intensities, a rectangle was selected and centered at the AP boundary across both ventral and dorsal compartments. Average pixel intensity was determined using the Plot Profile function of ImageJ and plotted using GraphPad Prism software.

Cells grown in glass-bottom dish were cultured in a chamber supplied with 5% CO₂ at 37 °C with humidity on the spinning disc microscope (Zeiss). Time lapse live images were taken with ZEN software under 40x oil lens for indicated time course.

After rehydration of slides, endogenous peroxidase activity was blocked by incubation in 1% H₂O₂/0.42% citric acid/1.08% Na₂HPO₄) and then boiled at 95 ±1 °C for 20 min in 40 mM Tris-HCl/64 mM EDTA pH9.0. Specimens were incubated in 1% BSA/PBS for 30 min followed by a 1-h to overnight incubation with the primary antibody in 0.05% BSA/PBS. After washing the slides briefly 2× in PBS and 1× in PBS-T (PBS/0.2% Tween) the samples were incubated for 45 min with EnVision+ (mouse or rabbit, DAKO) followed by color development in DAB substrate (Sigma) and mounted in Kaiser’s Glycerin-Gelatin for IHC. Incubation with Alexa488 and Alexa594-conjugated secondary antibodies was carried out for IF and slides were mounted with Citifluor AF1 antifadent solution (Citifluor limited)/DAPI.
Cells were plated on gelatin-coated cover slips for IF. After treatment they were washed in ice-cold PBS and fixed by either methanol or PFA treatment, −20 °C methanol for 15 min was used for membrane proteins and 4% PFA for 10 min followed by a 0.5% Triton-X100/PBS permeabilization step for 5 min for nuclear proteins. After blocking cells in 1% BSA they were incubated overnight in primary antibody solution diluted 1:200 in PBS. Secondary antibody incubation and mounting was done as described above. All IF samples were analyzed by confocal microscopy using a Zeiss Spinning disc microscope.

Cells were seeded in 24-well plates at a density of 10,000 cells per well and grown under standard culture conditions. After 24 h, the cells were transfected with the MitoTimer vector using the TransfeX kit in accordance with the manufacturer’s recommendations. Twenty-four hours after transfection, the cells were imaged by confocal microscopy (Zeiss spinning disc microscope) using an HC APO 63×/1.20 water objective. To ensure a constant molecular brightness of the MitoTimer, the following same settings (resolution: 512 × 512 format with a pixel size of 132 nm) were used:

Green fluorescence: excitation/emission—490/500–540 nm, EM gain—600, exposure time—750 ms, laser power—4%;

Red fluorescence: excitation/emission—550/580–640 nm, EM gain—700, exposure time—150 ms, laser power—4%.

Pictures were taken from randomly selected fields with a Z-stack section of 0.6 µm.
Quantification of the MitoTimer fluorescence was performed with ImageJ software. First, a mask was created covering all regions presenting green and/or red fluorescence, corresponding to the mitochondrial network. Furthermore, the mask was applied to extract mitochondria from the original image, the red and green fluorescence was quantified within the masked region, and the green-to-red fluorescence ratio was calculated. For each cell line at least 40 cells were analyzed.