Transfected immunostained neurons and live neurons in ACSF were imaged using a Nikon C2 inverted confocal microscope with a 60× oil immersion objective (1.4 numerical aperature, plan-Apo). Z-stack images were taken with 0.15 μm spacing at 1,024 × 1,024 resolution and parameters were kept constant between different conditions. Maximum intensity projections were generated using ImageJ Fiji software for all images except those using KDEL-CFP for which two individual stacks are shown (Schindelin et al., 2012 (link); Schneider et al., 2012 (link)).
C2 inverted confocal microscope
Manufactured by Nikon
The C2 inverted confocal microscope is a high-performance imaging system designed for advanced fluorescence microscopy applications. It features a inverted optical configuration, allowing for easy sample access and manipulation. The C2 utilizes a pinhole-based confocal architecture to provide optical sectioning and improved image contrast compared to conventional widefield microscopy.
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Lab products found in correlation
Plan apo, by Nikon (1 mentions)
Syto 9 green fluorescent nucleic acid stain, by Thermo Fisher Scientific (1 mentions)
Ula 96 well u bottom plate, by Corning (1 mentions)
Dulbecco modified eagle medium (dmem), by Roche (1 mentions)
Hbss buffer, by Corning (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Lonza (1 mentions)
X tremegene hp, by Roche (1 mentions)
5 protocols using c2 inverted confocal microscope
1
Confocal Microscopy of Neurons
2
Confocal Imaging of Neuronal Spines
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For confocal imaging of fixed neurons, Z-stack images (0.2 µm spacing, 1024 × 1024 pixel resolution) were acquired using a Nikon C2 inverted confocal microscope with a 60× oil immersion objective (1.4 NA, plan-Apo). Acquisition parameters (laser power, gain and offset) were kept constant between all conditions. Maximum intensity projections were generated using NIS Elements software and used for mask analysis of mean intensities for spine to shaft intensity ratio and colocalization for synaptic puncta.
3
Quantification of Biofilm Formation on Hydroxyapatite
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The in situ biofilms formed on the HAP discs were stained with 2.5 µM Syto 9 Green Fluorescent Nucleic Acid Stain (Invitrogen) for 15 min in dark as per manufacturer’s instructions. The discs were transferred to clean PBS in a glass-bottom plate, with biofilm facing towards the objective. Three-dimensional image stacks were collected from 5–6 fields of view selected randomly, with an inverted Nikon C2 inverted confocal microscope under 63X magnification. The data were quantified using Imaris 3D image processing software (Version 8.4, Bitplane, Oxford instruments, Zurich, Switzerland). Paired Student’s t-test was conducted using Minitab v.20, State College, PA USA, to compare the volume and thickness of the biofilms.
4
3D Spheroid Cytotoxicity Assay
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Approximately 5000 cells (2.5 × 104 cells/ml) cells were seeded in an Ultra-Low Attachment (ULA) 96-well U bottom-plate (Corning, NY, USA). Plates containing the cells were centrifuged at 210 Gav for 2 min to pellet the cells. Plates were incubated at 37 °C, 95% O2 , 5% CO2 for 72 h. After 72 h, 3D spheroids were embedded into collagen mix [18 ]. Spheroids were treated with A1331852, ABT-199 and S63845, alone and in combination for 72 h. Spheroid growth and invasion were photographed every 24 h using a Nikon C2+ inverted confocal microscope. Upon termination of the assay, live-dead staining of spheroids was conducted as described previously [19 ]. Images were taken using a Nikon-300 inverted fluorescence microscope. Growth of spheroids were analyzed using ImageJ (v1.51s, NIH) and statistical analysis (2-sided paired Student t tests) were performed using Microsoft® Excel. Using ImageJ (v1.51s, NIH), an outline was drawn around each spheroid in a focal Z plane which showed the maximum size and area and mean fluorescence was measured, along with adjacent background readings for control spheroids, spheroids treated with either S63845 or A1331852 and spheroids treated with combination of the two drugs. The total corrected red fluorescence (TCRF) = integrated density – (area of selected cell × mean fluorescence of background readings).
5
GPI-anchored Fluorescent Protein Variants
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U2OS cells were maintained in DMEM (Lonza) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified incubator at 37 °C, supplied with 5% CO2. A humanized cDNA encoding sGFPori fused to the GPI-anchoring domain of CD1414 (link) was used as template to generate different GPI-sFP variants by site-directed mutagenesis (Quickchange, Agilent Technologies) with appropriate primers. All the constructs were verified by DNA sequencing. Cells transiently transfected with the different GPI-sFP fusions (XtremeGENE HP, Roche) were imaged after incubation with 37 μM of M3 peptide in the DMEM + 10% FBS culture media for 12 hours. Cells were briefly rinsed with HBSS buffer (Corning) and imaged in the same buffer at 37 °C. Confocal fluorescence images were acquired on a Nikon C2 inverted confocal microscope equipped with a 488 nm excitation laser and a 525DF50 nm emission filter for imaging complemented GPI-sGFP variants and with a 515 nm excitation laser and a 542DF27 nm emission filter for imaging complemented GPI-sYFP variants.
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