Csu x

Images were taken using spinning disk microscope with a 100x Apo objective, NA 1.4 (Nikon Ti-E microscope equipped with Yokogawa spinning disk unit CSU-X and an Andor iXon 897 camera).
For lattice light sheet microscopy, 1 M cells were seeded in 6 well plates containing 5 mm cover glasses (#1.5 thickness) and transfected with either 0.1 μg pCDNA3.1-eGFP-SpRng2(1-189) or with 0.5 μg pEGFP-IQGAP1 (# 30112, Addgene) and 0.5 μg pTK93 Lifeact-mCherry. Cells were imaged 16-22 hours post transfection. Cover glasses were mounted on the imaging chamber and DMEM medium was replaced by pre-warmed L-15 imaging medium (cat. no. 11415049, Gibco, Fisher scientific). Imaging was done at 37°C on a 3i second generation lattice-light-sheet microscope with a 0.71 NA LWD WI objective for excitation and a 1.1 NA WI objective for imaging and equipped with 2 Hamamatsu ORCA-Flash 4.0v3 sCMOS cameras for simultaneous dual color imaging providing a 62.5x magnification with 230x230x370 nm (x-y-z) resolution using 488 nm and 561 nm lasers for excitation. 3D volumes were recorded with 0.57 μm step size for 150 planes with 100 ms exposure.

Cells or tissues were fixed in 4% paraformaldehyde (Electron Microscopy Services, 15710) in 1X PBS for 30 min at room temperature, washed and blocked with a blocking buffer (HBSS fortified with: 10% FBS (Hyclone), 0.1% BSA (Fischer, BP1600), 0.05% saponin (EMD, L3771), and 0.1% Tween 20 (Fischer, BP337500). Primary antibodies [1:100-1:200] for 2 h at RT or 24 h at 4 ºC, Secondary antibodies [1:1000] for 2 h at 22 ºC. Samples were imaged with a Nikon Eclipse Ti spinning disc microscope, Yokogawa CSU-X, Andor Zyla sCMOS, Andor Multi-Port Laser unit, and Molecular Devices MetaMorph imaging suite.
Antibodies used: Cleaved Caspase-3 (Asp175) (Cell Signaling, 9661, AB_2341188), HSF1 (Cell Signaling, 4356, AB_2120258), and HSP60 (LK1, sc-59567, AB_783870).