For laser microirradiation, virally transduced RPE1 cells expressing the indicated eGFP-tagged proteins were grown on glass coverslips and transfected with siRNAs. 48 h post-transfection, protein expression was induced using 0.5 µg/mL doxycycline, and 24 h later cells were presensitized with 1 μg/mL Hoechst for 15 min at 37 °C. DNA damage was introduced with a 355 nm laser (Coherent, Santa Clara, CA, USA, 40mW) focused through a Plan-Apochromat 40x oil objective to yield a spot size of 0.5-1 mm using a LSM780 confocal microscope (Zeiss) and the following laser settings: 100% power, 1 iteration, frame size 128 x 128, line step 7, pixel dwell: 25.21 μs.
Plan apochromat 40x oil objective
Manufactured by Zeiss
Sourced in United States
The Zeiss Plan-Apochromat 40x oil objective is a high-magnification microscope objective designed for detailed imaging. It offers a numerical aperture of 1.4 and a working distance of 0.21 mm, providing excellent optical performance and resolution. The objective is plan-apochromatic, ensuring flat field and chromatic aberration correction across a wide range of applications.
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4 protocols using plan apochromat 40x oil objective
1
Laser-Induced DNA Damage in RPE1 Cells
2
Confocal Microscopy Imaging of Whole Brain
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Tissues were mounted in Vectashield anti-fade reagent and imaged using a 3i Everest spinning disk confocal microscope. Whole brain images for Supplementary Fig 1 were taken using a Zeiss EC Plan-Neofluar 10X objective (NA=0.3). Whole brain images for Fig 4f were taken using a Plan-Apochromat 20X objective. All other images were taken using a Zeiss Plan-Apochromat 40X oil objective (NA=1.3). Images were stitched together when regions of interest did not fit within the field of view (Fig. 2a ).
3
Neurite Quantification in Primary Neuronal Cultures
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For neurite quantification, primary neuron cultures were stained for beta-tubulin III to outline neurites. 20 random fields of view were acquired for each replicate using a Plan Apochromat 40X Oil objective, with a 1.40 numerical aperture, with identical acquisition settings on a Zeiss Axio Observer 7, inverted, fluorescent microscope (Carl Zeiss Microscopy, LLC) and exported as original TIFFs using Zeiss ZEN 3.1 Blue Edition software (Carl Zeiss Microscopy, LLC). NeuriteTracer, an ImageJ plugin, was used to trace and measure neurites and count nuclei136 (link). The average neurite length for individual images was determined by dividing the total neurite length in an image by the total number of nuclei. To account for differences in neurite outgrowth between biological replicates, neurite length was normalized to hnRNP A1(WT)-transduced neurons within each replicate. Therefore, results are reported as a percentage of neurite length compared to hnRNP A1(WT).
4
Laser-Induced DNA Damage in RPE1 Cells
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For laser microirradiation, virally transduced RPE1 cells expressing the indicated eGFP-tagged proteins were grown on glass coverslips and transfected with siRNAs. 48 h post-transfection, protein expression was induced using 0.5 µg/mL doxycycline, and 24 h later cells were presensitized with 1 μg/mL Hoechst for 15 min at 37 °C. DNA damage was introduced with a 355 nm laser (Coherent, Santa Clara, CA, USA, 40mW) focused through a Plan-Apochromat 40x oil objective to yield a spot size of 0.5-1 mm using a LSM780 confocal microscope (Zeiss) and the following laser settings: 100% power, 1 iteration, frame size 128 x 128, line step 7, pixel dwell: 25.21 μs.
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