PC12 cells were rinsed with PBS and fixed in 4% paraformaldehyde in PBS and incubated for 20 min at room temperature. Cells were permeabilized in PBS containing 0.1% Triton X-100 for 10 min at room temperature and blocked in PBS containing 2% BSA, 1% fish skin gelatin, and 0.02% saponin. Primary antibodies were diluted in blocking solution at 1:1000 (SgII), 1:1000 (TGN38), 1:1000 (Syt1), 1:500 (HA)m and 1:500 (HA.11). The secondary goat anti-rabbit antibodies were diluted in blocking solution at 1:1000. Images were acquired using an Olympus Fluoview scanning confocal microscope and a 63× oil objective (NA 1.42) at a resolution of 512 × 512 pixels with a sampling speed of 12.5 μs/pixel with Kalman filter (integration count 5).
Na 1.42 oil objective
Manufactured by Olympus
The 60X NA=1.42 Oil Objective is a high-performance microscope objective designed for use in advanced scientific and research applications. It provides a magnification of 60X and a numerical aperture (NA) of 1.42, allowing for high-resolution imaging and detailed observations. The objective is optimized for use with oil immersion techniques, offering excellent optical performance and image quality.
Automatically generated - may contain errors
Lab products found in correlation
Fluoview scanning confocal microscope, by Olympus (2 mentions)
Deltavision deconvolution microscope, by Cytiva (1 mentions)
Softworx, by Cytiva (1 mentions)
Illustrator cs6, by Adobe (1 mentions)
Imagej, by Adobe (1 mentions)
Csu x1 spinning disk unit, by Yokogawa (1 mentions)
Orca flash 4.0 lt scmos camera, by Hamamatsu Photonics (1 mentions)
Ix71 inverted microscope, by Olympus (1 mentions)
5 protocols using na 1.42 oil objective
1
Immunofluorescence Imaging of PC12 Cells
2
Golgi-specific FRAP of HID-1 proteins
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INS-1 HID-1 KO cells stably expressing HID-1-GFP or G2A-GFP were transfected with sialyltransferase-pTAG-RFP657. The following day, transfected cells were plated on poly-L-lysine coated coverslips. Two days after transfection, coverslips were placed in an open imaging chamber (Thermofisher) and imaged using an Olympus Fluoview scanning confocal microscope and a 63× oil objective (NA 1.42) at a resolution of 512 × 512 pixels with a sampling speed of 8.0 μs/pixel with Kalman filter (integration count 5). The region of interest for photobleaching was selected based on proximity to the TGN and was positioned at the periphery of the cell. This was to ensure our primary objective of specifically photobleaching the cytosolic pool was met, while minimizing the risk of inadvertently affecting the Golgi-bound pool during the experiment. Cells were bleached for ~1sec at 100% laser output and imaged followed by a 15sec recovery period. Movies were taken for a total of 5min. Analysis of fluorescence intensity was restricted to the Golgi area by using the sialyltransferase-pTAG-RFP657 to generate an ROI within ImageJ.
3
Widefield Imaging of Fixed Actin Samples
4
Visualizing Microtubule Dynamics in HeLa Cells
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HeLa (7.5 × 104/well) cells were plated in 48 well-plates and after adherence were treated with ABZ (400, 800 and 1,600 nM), Paclitaxel (1 µM), Nocodazole (825 nM) or Noscapine (25 µM) for 12 h. The cells were fixed for 20 min (formaldehyde 4%, Triton X 0.5% in PBS). Cells were incubated overnight at 4°C with antibodies against α-tubulin (Sigma, 1:10,000) and crest (Cortex Biochem, 1:5,000) followed by 2 h of incubation with secondary antibody (Molecular probes, 1:500) and DAPI at room temperature. All antibodies were diluted in PBS/Tween 0.2%. The images were acquired using a Deltavision deconvolution microscope (Applied Precision) with a 60x (NA 1.42) oil objective (Olympus). Softworx (Applied Precision), ImageJ, Adobe Photoshop and Illustrator CS6 were used to process acquired images.
5
Spinning Disk Confocal Microscopy Imaging
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Images were acquired on an inverted IX71 microscope (Olympus) equipped with a CSU-X1 spinning disk unit (Yokogawa) and an iLas laser illumination system (Gataca Systems) with a 491 nm laser for illumination. A 60x NA 1.42 oil objective (Olympus) was used, and images were taken with an ORCA Flash 4.0LT sCMOS camera (Hamamatsu). The System was operated using the software MetaMorph.
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