Orcaflash 4lt camera

RNA in situ hybridization was performed as in Viswanathan et al. (2016) and Blice‐Baum AC et al. (in press, Aging Cell) with modifications: probes against Odd‐skipped and Gapdh1 were generated using the RNAscope (ACD) platform, and hybridized following the manufacturer's protocol. Labeled specimens were immediately mounted in Prolong Gold (Molecular Probes), and imaged the following day on an Imager.Z1 equipped with an OrcaFlash 4LT camera (Hamamatsu) and Apotome.2 (Zeiss), using ZEN software (2.3). 16‐bit dual channel images were analyzed using Fiji/ImageJ. Particles, representing individual messages, were quantified.

For images of higher magnification (including for colocalization of SQST-1::GFP and tdTOMATO::LGG-1/Atg8), animals were mounted on a 2% agarose pad in M9 medium containing 0.1% NaN₃ and imaged on a Zeiss Imager Z1 including apotome.2 with a Hamamatsu orca flash 4LT camera and Zen 2.3 software at ×1000 magnification. For colocalization, images were analyzed using the colocalization plugin Coloc2 in Fiji-ImageJ (National Institutes of Health). The correlation between the red and green fluorescence is given as intensity correlation quotient (ICQ) values^{68 (link)}. For random or mixed patterns of fluorescence intensity of the red and green channel this number will tend toward 0, for segregated localization of the red and green fluorescence signal the ICQ score will tend toward −0.5, and for colocalization of the fluorescent signals, the ICQ score will tend toward +0.5.

RoGFP was expressed in primary fibroblasts using modified pEGFP-N1 (RRID:Addgene_38120) as the expression vector and JetPei as the transfection reagent. After the cells were incubated in culture medium treated with or without HU or APH for 72 h at 37 °C, the cells were washed twice with Hanks’ balanced salt solution. For pEGFP-N1/roGFP1, the cells were imaged on a Zeiss Observer Z1 microscope with a Hamamatsu ORCA Flash 4LT camera. Images were acquired using MetaMorph software (Molecular Devices). For dual excitation ratio imaging, excitation filters at wavelengths of 400 nm and 488 nm were used, and an emission filter at a wavelength of 535 nm was used. The fluorescence excitation ratio was obtained by dividing the intensities of the cells using excitation filters at 400 nm and 488 nm.
For pEGFP-N1/roGFP-NLS (nuclear localization), the cells were incubated with DAPI (1 μg/ml) and imaged using a microscope. The images were captured using the 63x oil immersion objective of a motorized Axio Imager Z2 epifluorescence microscope (Carl Zeiss) equipped with a high-sensitivity cooled interline CCD camera (Cool SNAP HQ2; Roper Scientific) and a PIEZO stage (Physik Instrumente). Images were acquired using MetaMorph software (Molecular Devices). In each case, 300–500 cells were analyzed per condition.