Na oil immersion objective

To quantify the mitochondrial dynamics, time-lapse 3-D stacks for both cell types were acquired using Zeiss LSM 510 confocal microscope (Carl Zeiss Gmbh, Zena, Germany) equipped with environmental chamber, 63x/1.4NA oil immersion objective and a diode laser (excitation wavelength of 561 nm). The acquisition parameters were as followed: approx. 1% laser power for excitation of the mitoTracker Red CMX; zoom of 1.5 with line averaging of 2; pinhole of 1 Airy unit, X-Y pixel size of 0.09 μm, z-spacing of 1 μm, and a time step of 109 s. All images were 8-bit and covered an area of 1024×1024 pixels in the X-Y plane (∼84×84 μm), 4-6 z-sections (∼4–6 μm), and at least 8 time intervals (i.e., the experiment was ≥16 min).

Immunofluorescence microscopy was performed on cells fixed with 4% paraformaldehyde in PBS for 15 minutes at room temperature. Membranes were permeabilized using Triton X-100 (0.1%), followed by blocking with 1% BSA. Primary antibodies (diluted 1:100 in 1% BSA) were incubated for 1–2 hours at room temperature or overnight at 4 °C. Cells were then washed and incubated with secondary Alexa Fluor®-labeled antibodies (Life Technologies; diluted 1:150 in 1% BSA) for 1 hour at room temperature. DAPI and TRITC-phalloidin were used to stain nuclei and filamentous actin (F-actin). Images were acquired on the DV system described above, using a 60x/1.4 NA oil-immersion objective (Olympus), or a Zeiss LSM 880 laser scanning confocal microscope using a 63x/1.4 NA oil-immersion objective (Zeiss) or a 20x/0.8 NA objective (Zeiss).

Mutagenesis of GFAP (Origene, Rockville, MD, in vector CMV6-XL6) was performed using the QuikChange II mutagenesis kit (Agilent) to generate the designated point mutants. Sanger sequencing of the entire coding sequence of GFAP was performed to confirm the wild-type and mutant sequences. We used established procedures for the purification and in vitro assembly of GFAP (Perng et al., 2016 (link)). For transfections, lipofectamine 2000 was used according to the supplier instructions (Invitrogen, Thermo Fisher Scientific, Carlsbad, CA), and experiments were performed 20–24 hr after transfection. For immunofluorescence, cells were fixed in methanol at −20°C for 10 min, washed three times in PBS and incubated in blocking solution (2.5% bovine serum albumin, 2% normal goat serum in PBS) for 1 hr at room temperature. Primary antibodies were diluted into blocking buffer and incubated overnight at 4°C. The next day, cells were washed 3 times in PBS and incubated with Alexa Fluor-conjugated secondary antibodies diluted into blocking buffer for 1 hr at room temperature. Cells were washed 3 times in PBS, incubated in DAPI for 5 min, washed 3 times and mounted in Fluoromount-G (SouthernBiotech, Birmingham, AL) overnight. Cells were imaged on Zeiss 880 confocal laser scanning microscope using a 63x (1.4 NA) oil immersion objective (Zeiss, Jena, Germany).