Cells were washed with phosphate buffered saline (PBS) and fixed with pre-warmed 4% paraformaldehyde (PFA) for 10 min at room temperature. Coverslips were incubated with PBS with 3% (w/v) bovine serum albumin (BSA) and 0.1 % Triton X-100 for 20 min at room temperature to block and permeabilise neurons. Antibodies were mixed with PBS containing 3% (w/v) BSA to their appropriate working concentrations. Custom-made polyclonal anti-BoNT/A antibodies targeting the full-length toxin (Eurogentec) were diluted at 1:500, EEA1 antibodies (BD Biosciences, Cat. No. 610457) were diluted 1:200. These were incubated at 4 °C overnight. Coverslips were then washed and incubated with secondary antibodies (Jackson ImmunoResearch) in PBS with 3% (w/v) BSA for 1 h at room temperature. Coverslips were washed and mounted onto microscope slides using Fluoromount-G (Thermo Fischer Scientific, Cat. No 00-4959-52). A Leica SP5-AOBS confocal laser scanning microscope was used for confocal imaging.
Sp5 aobs confocal laser scanning microscope
Manufactured by Leica
Sourced in Germany, United Kingdom
The SP5-AOBS confocal laser scanning microscope is a high-performance imaging system designed for advanced microscopy applications. It features an acousto-optical beam splitter (AOBS) that enables flexible and efficient control of the excitation laser wavelengths, allowing for precise and tailored illumination of the sample. The SP5-AOBS provides a comprehensive set of tools for advanced imaging and analysis, catering to the needs of a wide range of research fields.
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Lab products found in correlation
Dmi6000 inverted epifluorescence microscope, by Leica (4 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (4 mentions)
Dmi6000 inverted microscope, by Leica (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Plan apochromat bl, by Leica (2 mentions)
Fetal calf serum (fcs), by Merck Group (2 mentions)
Fluoromount g, by Thermo Fisher Scientific (1 mentions)
Eea1 antibody, by BD (1 mentions)
Exo glow, by System Biosciences (1 mentions)
Huvecs, by Lonza (1 mentions)
Volocity 3d image software, by PerkinElmer (1 mentions)
Rabbit anti calbindin d28k, by Merck Group (1 mentions)
Alexa fluor 555 goat anti rat, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 555 goat anti mouse, by Thermo Fisher Scientific (1 mentions)
H 1200, by Vector Laboratories (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
Slowfade reagent, by Thermo Fisher Scientific (1 mentions)
1 4 diazabicyclo 2.2.2 octane, by Merck Group (1 mentions)
Mowiol, by Merck Group (1 mentions)
17 protocols using sp5 aobs confocal laser scanning microscope
1
Immunofluorescence Visualization of BoNT/A
2
Confocal Imaging of Fixed Cells
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Cells were prepared for confocal immunofluorescence microscopy by fixation in 4% paraformaldehyde. Confocal microscopy was performed using a Leica SP5 AOBS confocal laser-scanning microscope with an attached Leica DM I6000 inverted microscope. Confocal sections were taken across the z-plane and processed to form a 2D projection representing the full depth of the cell culture.
3
Exosome Uptake in Endothelial Cells
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PF-derived exosomes were labeled using Exo-Glow based on carboxyfluorescein succinimidyl diacetate ester (CFSE) chemistry (System Biosciences) according to the manufacturer’s recommendations. Human umbilical vein ECs (HUVECs) (Lonza) were seeded at a density of 5 × 104 cells/well on a 24-well plate coverslip, and 10 μg/ml of labeled PF-derived exosomes were added to target ECs in culture for 24 hr at 37°C. Cells were washed twice with PBS and fixed with 4% buffered PFA (Sigma) in PBS for 20 min at RT. Nuclei were stained by DAPI staining while actin filaments were labeled using Rhodamine Phalloidin (Thermo Fisher Scientific). To assess the uptake of exosomes by ECs, confocal images were acquired with a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope. All images were collected using a 63× NA 1.4 oil immersion lens objective. The excitation signals for Exo-Glow and Rhodamine Phalloidin were 494 and 540 nm, respectively. The fluorescence emitted from the cells was recorded at 521 nm for Exo-Glow and 565 nm for Rhodamine Phalloidin. In all cases, z-stack images were obtained covering the entire cell volume. Three-dimensional reconstruction of the confocal image z stacks confirmed the cytoplasmatic localization of internalized exosomes.
4
Quantitative Immunofluorescence Imaging of Purkinje Cells
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Sections were deparaffinised, hydrated and washed as above. An antigen retrieval step was performed through microwaving in sodium citrate buffer (0.01 M, pH 6.0, 5 min). Purkinje cells were labelled by single or double immunofluorescence using rabbit anti-Calbindin-D28K (1:500) (Sigma-Aldrich, UK), mouse anti-SMI-34 (phosphorylated neurofilament; 1:500) (Covance, US) and rat anti-myelin basic protein (MBP) (1:100) (Serotec, UK). Non-specific binding was blocked with 10 % normal goat serum diluted in PBS containing 0.1 % triton. Sections were incubated at 4 °C overnight with primary antibodies. Sections were then washed in PBS and incubated for 30 min in the dark with Alexa Fluor 555, goat anti-mouse (1:500), Alexa Fluor 488, goat anti-rabbit (1:500) or Alexa Fluor 555, goat anti-rat (1:500) (Invitrogen, Paisley, UK), before being washed in PBS and mounted in Vectashield medium containing the nuclear dye 4′6′-diamidino-2-phenylindole (DAPI) (H-1200, Vector Laboratories). Sections were imaged using either: 1) a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope with Leica Application Suite Advanced Fluorescence software and Volocity 3D image software (PerkinElmer, USA); or 2) a Nikon C1 confocal microscope and EZ viewer software.
5
FRET Analysis of Protein Interactions
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FRET analysis has been performed as previously described [35 (link)]. Briefly, U-2 OS cells cultured on glass coverslips were transfected with constructs expressing CFP- and YFP-tagged proteins and fixed at 24 h posttransfection with 2% paraformaldehyde for 5 min and the coverslips were mounted on glass slides with SlowFade reagent (Thermo Fischer Scientific). Acceptor photobleaching FRET was performed using an SP5 AOBS confocal laser scanning microscope (Leica; Wetzlar, Germany).
6
Confocal Imaging of Transfected Neurons
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Confocal imaging was performed using a Leica SP5-AOBS confocal laser scanning microscope linked to a Leica DMI 6000 inverted epifluorescence microscope with the laser lines: 405 (blue for the nucleus), 488 (green for the total), 633 (far red for the surface). The transfected neurons were imaged by looking for the total labelling of GluK2 (green cells). A 63x oil immersion lens was used for image acquisition. Each image is composed of 5–6 stacks (0.4–0.5 μm stack interval) that were projected by maximum intensity. The untreated WT GluK2 condition was used to optimise the settings, which were kept constant throughout the same experiment. The immunofluorescence was quantified using ImageJ (FIJI).
7
Quantifying Secreted Growth Factors
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The concentration of VEGF-A, bFGF and PDGF-BB was directly measured in culture supernatants using enzyme-linked immunosorbent assay (ELISA) kits following the manufacturer’s recommendations. For cell line measurements R&D Systems (Minneapolis, MN, USA) and for tumoroids Thermo Fisher Scientific (VEGF and bFGF) and R&D Systems (PDGF-BB). Tumoroids were cultured in the CACC assay and supernatants collected on day 11. VEGF-expressing SW480 cells (SW480-V) and the parental line were cultured to confluence and allowed to condition the cell media for 48h. VEGF secretion was also examined qualitatively by immunofluorescence microscopy. Briefly, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 5 min. Cells were incubated with VEGF antibody (R&D systems) for 1h, followed by secondary Alexa-488 conjugated goat anti-mouse antibody (Invitrogen Carlsbad, CA, USA) for 45 min. Accordingly, the cells were incubated with DAPI for 5 min and mounted in Mowiol (Sigma-Aldrich) containing 2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO) as an anti-bleaching agent. Images were obtained using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope.
8
Cell Preparation for Immunofluorescence Microscopy
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The preparation of cells for immunofluorescence microscopy was carried out as previously described (19) . Briefly, cells were fixed in 4% paraformaldehyde in PBS for 15min. The cells were then permeabilized with 0.2% Triton X-100 in PBS for 5min. Cells were then treated with 0.1% freshly-prepared sodium borohydride in PBS for 5min to quench autofluorescence. The cells were washed three times in PBS between each step. The fixed cells were incubated with primary antibodies for 1h in PBS, followed by incubation in secondary antibodies for 45min.
Cells were washed three times in PBS between each step. The cells were then incubated with 300nM DAPI for 10min and mounted under Mowiol containing 2.5% (w/v) DABCO as an antifade agent. Confocal microscopy was performed using a Leica SP5 AOBS confocal laserscanning microscope with an attached Leica DM I6000 inverted microscope. Confocal sections were taken across the z-plane and processed to form a 2D projection representing the full depth of the cells.
Cells were washed three times in PBS between each step. The cells were then incubated with 300nM DAPI for 10min and mounted under Mowiol containing 2.5% (w/v) DABCO as an antifade agent. Confocal microscopy was performed using a Leica SP5 AOBS confocal laserscanning microscope with an attached Leica DM I6000 inverted microscope. Confocal sections were taken across the z-plane and processed to form a 2D projection representing the full depth of the cells.
9
Immunohistochemical Analysis of Mouse Retina
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Eyes from euthanised mice were enucleated and fixed in 4% v/v PFA for 1 h before dissection. The anterior portion of the eye (cornea, iris, ciliary body, and lens) was carefully removed and an eyecup prepared. The eyecup tissue was blocked in 5% v/v normal goat serum (Vector Laboratories, CA, USA), 1% v/v BSA, and 3% v/v Triton x-100 (both Sigma Aldrich) in PBS for 4 h at room temperature with gentle shaking. Eyecups were then incubated at 4°C overnight with a rabbit anti-mouse anti-RFP mAb (600-401-379; Rockland Immunochemicals Inc., Limerick, PA) and for target validation experiments a Super Bright 600-conjugated anti-mouse CD44 mAb (Supplementary Table 1 ) was used in combination. After thorough washing with PBS, samples were incubated overnight with the secondary antibody goat anti-rabbit Alexa-633 (A21070; Thermo Fisher Scientific). The eyecups were washed again, and the retina carefully removed and flatmounted in Vectashield hard-set antifade mounting media (H-1400; Vector Laboratories Ltd., Peterborough, UK) and imaged on a Leica SP5-AOBS confocal laser scanning microscope (Leica Microsystems Ltd., Wetzlar, Germany). Images were acquired with an xy pixel size ≤ 200 nm, and a z-step size of ≤ 400 nm.
10
Immunofluorescence Microscopy of HeLa Cells
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HeLa cells were seeded on coverslips in 6-well plates prior to transfection. 24 h after transfection, cells were fixed with 4% formaldehyde (Thermo Scientific) for 15 min or icecold methanol for 5min. Fixed cells were blocked in 1% BSA and 4% FCS (Sigma-Aldrich) buffer and incubated for 30 min with primary and secondary antibodies (listed above) diluted in blocking buffer. Cells were DAPI (diamidino-2-phenylindole, Thermo Scientific) stained and placed cell side down in FluorSave reagent (Calbiochem). Confocal microscopy was carried out using a Leica SP5-AOBS confocal laser scanning microscope attached to a Leica DM I6000 inverted epifluorescence microscope (Leica Microsystems, Heidelberg, Germany). A 63x 1.4 NA oil immersion objective (Plan Apochromat BL; Leica Biosystems) and the standard SP5 system acquisition software and detector were used.
Image analysis was performed using ImageJ. For co-localisation studies, Pearson's correlation coefficient was calculated from ∼ 40 cells per condition using ImageJ.
Image analysis was performed using ImageJ. For co-localisation studies, Pearson's correlation coefficient was calculated from ∼ 40 cells per condition using ImageJ.
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