T cells (Jurkat or SAg‐T blasts) alone or in conjugates with Raji cells were seeded onto coverslips precoated with poly‐L‐lysine (Sigma) prior to fixation with 4% PFA for 10 min at RT. After three washings with PBS, the coverslips were then incubated with NH4Cl for 10 min to remove residual PFA. TCR and F‐actin were stained with anti‐CD3 Ab (clone UCHT1, Biolegend) and Phalloidin AF647 (ThermoFisher, A22287), respectively, followed by staining with secondary antibodies for 45 min at RT. Depending on the Abs, several protocols were used for intracellular staining. For mouse anti‐human Lck, permeabilisation with PBS supplemented with 0.05% saponin was performed for 15 min at RT, followed by incubation with the Ab diluted in the same buffer for 1 hr. For rabbit‐anti human‐GM130 (Abcam, ab52649) and mouse‐anti‐Rab11 (BD Bioscience, 610,657), permeabilisation was done with PBS supplemented with 0.1% Triton. Confocal microscopy was performed on a SP5 (Leica) or on an Opterra (Bruker) using either a 63× objective or a 100× objective (NA 1.4). Z‐stack acquisitions of optical sections were performed at 0.3 increments for analysis. Images were analysed using Fiji analysis software (Schindelin et al., 2012 ).
Opterra
Manufactured by Bruker
The Opterra is a multiphoton microscope system designed for high-speed, high-resolution imaging of living specimens. It utilizes a resonant scanning system to achieve fast image acquisition rates, making it suitable for applications that require real-time observation of dynamic biological processes. The Opterra provides researchers with a tool for capturing detailed, three-dimensional information about the structure and function of living cells and tissues.
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Lab products found in correlation
3 protocols using opterra
1
Visualizing T-Cell Receptor and Actin Dynamics
2
Multicolor Live Cell Imaging
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HEK293T cells or THP-1 cells were grown on the eight-well chamber (Nunc). Cells were labeled with Mitotracker Red, Mitotracker Deep Red FM, TubulinTracker Green, and Hoechst following manufacturer’s instruction. Nucleus was revealed in blue in all the images. Live cell imaging was performed at 37 °C using confocal microscopes: Carl Zeiss LSM 700, Opterra (Bruker) or Andor Spinning Disk (Oxford Instruments). Z-sections were taken to reconstitute 3D structure. Images were processed and analysed using ImageJ.
3
Histological Verification of In Vivo Recordings
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Immediately upon completion of a successful recording, animals were deeply anaesthetised with an overdose of ketamine/xylazine administered intraperitoneally and promptly perfused transcardially with 1× phosphate-buffered saline followed by 4% paraformaldehyde solution. Brains were extracted and kept immersed overnight in 4% paraformaldehyde solution. 60–70 μm-thick coronal slices were prepared from the recorded hippocampi. Slices were stained with Alexa Fluor 488–streptavidin to reveal biocytin-filled neurons and patch electrode tracts. DAPI was applied as a nuclear stain to reveal the general anatomy of the preparation. Fluorescence images were acquired using a spinning-disc confocal microscope (Opterra, Bruker) and analysed using ImageJ. The accuracy of the recording coordinates was confirmed in all cases by identification of either the recorded neuron or the recording electrode tract.
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