Caco-2 cells were seeded and cultured in FluoroDish cell culture dishes as described above. Cells were rinsed twice with D-PBS (Life Technologies) and incubated with 1000 μL of either 1 µM or 5 µM TAMRA-labeled peptides in OptiMEM (Life Technologies) for 3 hours at 37°C and 5 % CO2. 10 min before the end of the incubation, 1 µg/mL Hoechst 33342 (Sigma-Aldrich) and 1 µg/mL WGA-Alexa488 (Invitrogen) were added for nuclei and membrane staining, respectively. The incubation medium was then removed, cells were rinsed twice with D-PBS and covered with 1.5 mL of FluoroBrite (Life Technologies). Fluorescence was observed at 37°C with a Leica SP5-SMD confocal microscope (Leica HCX PL Apo CS 63×/1.4NA oil objective lens; Hoechst 33342: λex = 405 nm / λem = 415–485 nm; TAMRA: λex = 561 nm / λem = 571-nm; Alexa488: λex = 488 nm / λem = 498–548 nm). Image acquisition was done sequentially to minimize crosstalk between the fluorophores. Each confocal image was merged and adjusted with the same brightness and contrast parameters using the ImageJ software.
Sp5 smd confocal microscope
Manufactured by Leica
The SP5-SMD confocal microscope is a high-performance imaging system designed for advanced microscopy applications. It features a scanning confocal architecture that provides optical sectioning and improved image contrast. The system is capable of capturing detailed, high-resolution images of samples, enabling researchers to study various specimens with precision and clarity.
Automatically generated - may contain errors
Lab products found in correlation
Wga alexa488, by Thermo Fisher Scientific (1 mentions)
Dulbecco phosphate buffered saline (dpbs), by Thermo Fisher Scientific (1 mentions)
Opti mem, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Merck Group (1 mentions)
Hoechst 33342, by Leica (1 mentions)
Alexa 488, by Leica (1 mentions)
Lysotracker red, by Thermo Fisher Scientific (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Axio vert a1, by Zeiss (1 mentions)
Observer z1, by Zeiss (1 mentions)
Slowfade antifade reagent, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 or 546 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Taxol, by Merck Group (1 mentions)
Mouse anti lys40 acetylated alpha tubulin, by Merck Group (1 mentions)
8 protocols using sp5 smd confocal microscope
1
Visualizing Peptide Internalization in Caco-2 Cells
2
Live-Cell Organelle Imaging Protocol
3
Immunofluorescence Imaging of Transfected Cells
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The transfected cells and controls were fixed with 4% paraformaldehyde (PFA) and stained with anti-hemagglutinin tag primary antibodies followed by corresponding secondary antibodies and Hoechst 33342. Cells fixed on the glass coverslips were imaged using Leica SP5 SMD confocal microscope, equipped with 60 × HCX PL APO CS NA 1.4 lens. Excitation was 488 nm (for Alexa 488) and 405 nm (for Hoechst). Emission was recorded in 415–460 nm and 500–535 nm ranges. Images shown in Fig. 2B were histogram stretched.
4
Immunostaining of Osteoclasts for GEF-H1 and Cytoskeleton
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Osteoclasts on 13-mm diameter glass or ACC-coated coverslips were either fixed for 20 min in 3.2% paraformaldehyde in PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO4, pH 6.9), permeabilized with 0.1% Triton X100 for 1 min and blocked with 1% BSA in PBS for 15 min or permeabilized and blocked with PBS - 2% BSA - 0.2% triton X100 for 1 h (GEF-H1 staining). Then osteoclasts were incubated for 1 h with primary antibodies: rabbit anti-GEF-H1 (Abcam, #ab155785, 1/200), mouse anti-alpha tubulin (Sigma, #T-5168, 1/2000) and/or Alexa 564-phalloidin (Life Technologies, #A22283, 1/1000). Signal was revealed with the adapted Alexa Fluor 488-, 546 or 647-conjugated secondary antibodies (Life Technologies, 1/1000). Preparations were mounted in Citifluor mounting medium (Biovalley) and imaged with Leica SP5-SMD confocal microscope using 40X HCX Plan Apo CS oil 1.3NA or 63X HCX Plan Apo CS oil 1.4NA objectives. Co-localization was shown using ImageJ pluggin DiAna (Gilles et al., 2017 (link)).
5
Fluorescence Microscopy Imaging Protocol
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Two days after transfection, epifluorescence images were acquired using a Zeiss Observer.Z1 inverted microscope equipped with a × 20 Plan-Neofluar objective or a Zeiss Axio Vert.A1 inverted microscope equipped with a × 40 Plan-Neofluar objective. Confocal microscopy was performed on a Leica SP5 SMD confocal microscope with a × 63 water-immersion objective.
6
Neuronal Stimulation FCS Imaging
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All FCS recordings were acquired using a Leica SP5 SMD confocal microscope using a × 63 1.2NA HCX PL Apo water lens and 488 or 561 nm CW lasers. Photon fluctuation data routed through a Picoquant PRT 400 router were acquired at microsecond rates using external Single Photon Avalanche Photodiodes (MicroPhoton Devices, Italy). Neurons were placed in a custom-made chamber with embedded platinum wires to deliver a stimulus. Cells undergoing electrical stimulation were immersed in stimulation buffer (136 mM NaCl, 2.5 mM KCl, 10 mM glucose, 10 mM HEPES (pH 7.4), 2 mM CaCl2, 1.3 mM MgCl2) and stimulated at 20 Hz for 30 s allowing all FCS data to be acquired during the stimulation train.
7
Immunofluorescence Staining and Confocal Microscopy
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Cells were plated on poly-D-lysine coated coverslips, fixed with 4% PFA (Alfa Aesar) for 10 min, permeabilized with 0.1% Triton X100 for 15 min, neutralized with 50 mM NH4Cl for 10 min, and blocked with 2% BSA for 10 min. Cells were incubated with primary and secondary antibodies for 1 h and 45 min, respectively, at room temperature in a wet chamber. Finally, cells were labelled with Hoescht and mounted in SlowFade antifade reagent (Thermo Fischer Scientific). Images were acquired with a Leica SP5-SMD confocal microscope. Mander’s coefficients were determined by counting 3 fields of around 300 cells per condition using the JAcoP plugin (ImageJ).
8
Immunofluorescent Imaging of Osteoclasts
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Osteoclasts differentiated on 13 mm-diameter glass coverslips were fixed for 20 min, in 10 µM Taxol (Sigma, #T7402) and 3,2% paraformaldehyde in PHEM (60 mM Pipes, 25 mM Hepes, 10 mM EGTA, 4 mM MgSO 4 , pH 6.9). After permeabilization with 0.1% Triton X100 for 1 min and blocking with 1% BSA in PBS for 15 min, osteoclasts were incubated for 1 h with Alexa Fluor 647-labelled phalloidin (1/1000, Life Technologies, #A22287), mouse anti-lys40 acetylated alpha tubulin (1/500, Sigma, #T6793), rat anti-alpha tubulin (1/200, Santa Cruz, #sc-53030) or rabbit anti-Dock5 (1/100, homemade) and revealed with the adapted Alexa Fluor 488 or 546-conjugated secondary antibodies (1/1000, Life Technologies). Preparations were mounted in Citifluor mouting medium (Biovalley) and imaged with Leica SP5-SMD confocal microscope using 40X HCX Plan Apo CS oil 1.3NA or 63X HCX Plan Apo CS oil 1.4NA objectives. All imaging was performed at the Montpellier RIO Imaging facility (http://www.mri.cnrs.fr/en/). Immunofluorescent signals integrated density was quantified using ImageJ.
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