Lsm 510 inverted microscope

Acellular HDF-derived ECM and collagen I substrates were prepared. Keratinocytes were harvested and seeded on these substrates at a density of 1 × 10⁴ cells/well in a 24-well tissue culture plate (NUNC) and grown for four days in DKSFM with medium changes on alternate days. After 4 days, the culture medium was replaced with DMEM/Ham’s F12 supplemented with 2% human serum (Sigma), and the cells cultured for an additional 24 hrs. Keratinocyte sheets were fixed, permeabilised with 0.1% Triton-X100 in PBS, blocked with BSA/1% goat serum/PBS, and stained with 20 μg/ml of anti-E Cadherin mAb, 4A2C7 (Zymed, CA, USA), anti-mouse Alexa 488 conjugated antibody and DAPI, as detailed in immunocytochemistry analysis. The keratinocyte sheets were detached from their substrates by incubating at 37 °C for 30 min with 2.4 U/ml dispase (Roche Diagnostics, Basel, Switzerland) in DMEM/Ham’s F12 supplemented with 10 mM HEPES (Gibco). Detached sheets were washed with DMEM/Ham’s F12/HEPES and transferred to a 10 ml tube containing DMEM/Ham’s F12/HEPES. To assess the strength of the keratinocyte sheets, the 10 ml tubes underwent 60 inversion cycles (one inversion cycle/sec) on a rocker before being transferred to a 24-well plate for imaging. Keratinocyte sheets were imaged using a Zeiss LSM510 inverted microscope.

For confocal microscopy imaging of live cells, 1.8 × 10⁵ cells seeded onto 24-mm round glass coverslips transfected and treated as indicated were incubated in Hank’s Balanced Salt Solution (HBSS) supplemented with 10 mM HEPES and coverslips were placed on the stage of a Zeiss LSM 510 inverted microscope. Cells expressing mtYFP and mtRFP were excited using the 488 nm or 543 nm line of the Argon laser using a 63c × c1.4 NA Plan Apochromat objective (Zeiss).
For the mitochondrial docking assay (Ishihara et al., 2004 (link)), mitochondria from cells of the indicated genotype expressing mtRFP or mtYFP were isolated and resuspended in EB (0.5 mg/ml) at 25°C for 30 min with 1 mM GTP unless indicated. Mitochondria expressing mtRFP or mtYFP were mixed in 1:1 ratio, centrifuged, and incubated for 15 min. Mixed mitochondria were mounted on a 24-mm round coverslip, allowed to attach for 15 min, placed on the stage of a Leica SP5 confocal inverted microscope and excited using the 488 nm or 543 nm line of an Ar laser using a 63c × c1.4 NA Plan Apochromat objective (Leica).

Confocal immunofluorescence microscopy experiments were accomplished as previously described by us [48 (link)]. Briefly, cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min before being permeabilized through 5 minute-treatment with 0.1% Triton X-100. Cells were then washed with PBS (pH 7.2) and blocked with 0.5% BSA in PBS before being probed with primary antibodies and subsequent secondary fluorophore-conjugated antiserum (Alexa Fluor 488 and 564). Anti-FLAG antibody (M2, Sigma) was used at 1:1000, anti-FLAG polyclonal antibody; 1:400, anti-Myc 9E10; 1:100, anti-Sec31A; 1:100, anti-ERGIC-53; 1:100, anti-GM130; 1:100, anti-EEA1; 1:100, anti-Sec16A; 1:200, and anti-MAVS; 1:100. Secondary fluorophore-conjugated antiserum (Alexa Fluor 488 and 564) was used at 1:500 in PBS 0.5% BSA. The nucleus was labeled by 4′,6-diamidino-2-phenylindole (DAPI) staining. The confocal micrographs represent a single optical section (Z-stack) of cells. Images were acquired from a LSM 510 inverted microscope (Zeiss) combined to LSM v3.2 software (Zeiss). Colocalization of labeled protein was assessed by linescan analysis using “Profile” function in the ZEN 3.1 blue software (Zeiss). The pixel intensity in each channel is measured along a line drawn on the image and is plotted versus distance along the line.