For AVA [left (L) or right (R)] and AVB (L or R) co-imaging, an individual transgenic L3-stage animal expressing the GCaMP6s calcium sensor in AVA and AVB (hpIs819) was mounted on a 2% NGM gel pad with ~1 μl of M9 buffer so that it could move under a coverslip. Mounted animals were allowed to recover for 3 min on the pad before recording. The recording was performed using a 20× objective on Nikon spinning disc confocal microscope. Animals were centered in the field of view by manual tracking. AVB soma was manually kept in focus at the RFP channel during recording. The initiation of each epoch of backward to forward locomotion transition was defined by the alignment between velocity and AVA-AVB calcium activity (GCaMP6s intensity) using in-house–developed MATLAB scripts (82 (link)).
Spinning disc confocal microscope
Manufactured by Nikon
Sourced in Canada
The Spinning disc confocal microscope is a type of laboratory equipment used for high-resolution imaging of specimens. It utilizes a spinning disc with multiple pinholes to rapidly scan and illuminate the sample, allowing for the capture of images with improved optical sectioning and reduced background noise compared to traditional microscopy techniques.
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Lab products found in correlation
Hoechst 33342, by Thermo Fisher Scientific (4 mentions)
Widefield epifluorescent microscope, by Nikon (2 mentions)
Alexa fluor 488 donkey anti rabbit, by Thermo Fisher Scientific (2 mentions)
Anti phospho her3 tyr1289, by Cell Signaling Technology (2 mentions)
Anti met d1c2, by Cell Signaling Technology (2 mentions)
Anti phospho met tyr1234 y1235, by Cell Signaling Technology (2 mentions)
Anti phospho egf receptor tyr1068, by Cell Signaling Technology (2 mentions)
Anti golgin 97, by Thermo Fisher Scientific (2 mentions)
Anti pdi rl90, by Thermo Fisher Scientific (2 mentions)
Anti her3 d22c5 xp, by Cell Signaling Technology (2 mentions)
Alexa fluor 568 goat anti mouse, by Thermo Fisher Scientific (2 mentions)
Anti flag m2 antibody, by Merck Group (2 mentions)
Anti eea1, by BD (2 mentions)
Cellmask orange plasma membrane stain, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
R10367, by Roche (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Nis elements ar software, by Nikon (1 mentions)
29 protocols using spinning disc confocal microscope
1
Calcium Imaging of AVA and AVB Neurons
2
Imaging Subcellular Localization of TWK-40
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Mixed stages of animals with endogenously tagged twk-40 (twk-40::TagRFP::ZF), with or without the plasma marker (Prgef-1-GPI::YFP), and animals with an integrated twk-40::GFP fosmid were fixed in 2% paraformaldehyde at 4°C for 1 hour and processed as previously described (102 (link)). Primary antibodies against TagRFP (Thermo Fisher Scientific, R10367, rabbit) and/or green fluorescent protein (GFP) (Roche, mouse) were used at 1:500 and 1:200 dilution, respectively. Secondary antibodies of goat–anti-rabbit (Alexa Fluor 488) and/or goat anti-mouse (Alexa Fluor 594) were used at 1:5000 dilution. L4 larvae and adults were imaged with a Nikon spinning disc confocal microscope with a 63× objective and reconstructed by maximum intensity projection. Single layers of acquired images from animals co-expressing TWK-40::TagRFP::ZF and GPI::YFP were examined for subcellular colocalization using an ImageJ plugin (JACoP).
3
Measuring Synaptopodin Dynamics in Neurons
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Primary hippocampal neurons were co-infected with two AAVs containing GFP–synaptopodin and mRuby2. mRuby2 was imaged with a 563 nm laser and the images were used to determine the localization of synaptopodin puncta. FRAP of GFP–synaptopodin was performed using a Nikon spinning disc confocal microscope. Time-lapse imaging of GFP–synaptopodin was performed with a 488 nm laser and bleaching of selected puncta was achieved with a 405 nm laser. Images were acquired at 0.5 Hz for 300 s, starting with a five frame baseline before FRAP. FRAP analysis was undertaken using the plug in Time Series Analyzer V3 of Fiji (NIH, Bethesda, MD, USA). Regions of interest (ROIs) were selected on the bleached (‘frapped’) synaptopodin puncta, on a non-bleached stretch of dendrite to account for bleaching during imaging (bleach control) and on a non-fluorescent region to account for background fluctuations. The integrated density values of those ROIs were measured, the background values were subtracted from the values of GFP–synaptopodin puncta, then GFP–synaptopodin values were normalized to the bleach control and to the pre-bleach value (i.e. the pre-beach value was considered as 100%).
4
Imaging Microtubule and Mitochondria Dynamics
5
Immunofluorescence Imaging of Frozen Cardiac Tissues
6
Pathogen-induced cell death kinetics
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BMNs were seeded into glass bottom 96-well plates at a density of 2.5 × 105 cells/well. Overnight (16h) primed BMNs were infected with P. aeruginosa at an MOI of 10 for 60, 120, or 180 minutes. DNA was detected by cell impermeable DNA fluorescent dye (2.5μΜ Sytox Green) along with cell-permeable DNA dye (1μg/ml Hoechst). Dyes were added 30 minutes before imaging. Fluorescent images were taken with a 40x objective with a Nikon spinning disc confocal microscope. Images were processed and analyzed using the Nikon NIS-Elements AR software (version 5.42.02).
7
Quantifying Receptor Localization and Activation
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9×104 COS7 cells were plated on coverslips. After 24 hours, cells were transfected and cultured for 24–48 hours. Cells were fixed with 3.7% formaldehyde for 1 hour at room temperature, permeabilized with 0.1% Triton X-100 for 5 minutes, and blocked with 1% BSA for 5 minutes. Cells were incubated with primary antibodies at 37C for 1–2 hours: anti-FLAG-M2 antibody (Sigma Aldrich #F1804), anti-phospho-HER3 Tyr1289 (21D3 – Cell Signaling), anti-MET (D1C2 – Cell Signaling), anti-phospho-MET Tyr1234/Y1235 (Cell Signaling), anti-phospho-EGF-Receptor Tyr1068 (Cell Signaling), anti-Golgin97 (A-21270 – Molecular Probes), anti-PDI (RL90 - Thermo Scientific), or anti-EEA1 (BD Biosciences - 610456), anti-HER3 (D22C5 XP – Cell Signaling). Cells were washed and incubated with fluorescently tagged anti-rabbit or anti-mouse secondary antibodies (Goat Anti-Mouse Alexa-fluor-568 cat#A11031; Alexa-Fluor-488 donkey anti-rabbit cat# A21206 – Life technologies). Images were taken at 60x magnification using a Nikon widefield epifluorescent microscope (Fig. 3a , b , 5a -d , 7b ), or at 60x using a Nikon spinning disc confocal microscope Confocal images shown were taken as a stack of 7 slices in the Z-plane and processed as a Z-max projection (Supplementary Fig. 4 ).
8
Quantifying Receptor Localization and Activation
9
Imaging TG2 Inhibitor and Mitochondria
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SCC-13 and HaCaT cells (0.1 × 106) were plated in 35 mm Mat Tek glass bottom cell culture dishes. After 24 h, the cells were treated with 25 µM 18 (NCEG-RHB ), a rhodamine-B-labelled cell-permeable TG2 inhibitor, for 18 h. The cells were washed three times with phosphate-buffered saline prior to spinning disc confocal microscopy. In addition, the HaCaT cells were treated with 50 nM MitoTracker GreenFM (#M7514) dye, obtained from Invitrogen (Waltham, MA, USA). The cells were then washed three times with PBS before live cell imaging using a Nikon spinning disc confocal microscope. HaCaT and SCC-13 are epidermis-derived cutaneous squamous cell carcinoma cells [59 (link),60 (link)]. NCEG-RHB (18 ) is detected adjacent to the nuclei (N ) in both cell types and is indicated by red arrows. MitoTracker GreenFM stains mitochondria membrane proteins inside the cell, which is indicated by the green arrows. The sizing bar in the images represents 100 microns.
10
Paternal Pronucleus Morphometrics from Spinning Disc Confocal
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Images captured on the Nikon spinning disc confocal microscope were converted into maximum projections with FIJI. Videos were saved as image sequences (.jpg) and read by the MATLAB script. Paternal pronuclei were segmented using a fluorescent threshold to transform the images into binary. Nuclear diameter was calculated using the MALTAB function ‘regionprops’ for each time frame. The surface area (formula) and volume (4/3×pi×r^3) were calculated using the radius for the paternal pronucleus.
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