Experiments were performed essentially as previously described [26] (link). In brief, islets were loaded with 10 μM Fluo-8 AM (Abcam) dissolved in DMSO (0.01% wt/vol) in Krebs-HEPES buffer for 45 min at 37 °C. Islets were visualised using a Zeiss Axiovert confocal microscope and perifused continuously at 34–36 °C with Krebs-HEPES buffer containing relevant concentrations of glucose and GLP-1. Fluo-8 was excited with a 491 nm laser and emitted light collected at 525 nm. Volocity™ software (Perkin–Elmer) was used for imaging and analysis. Traces were normalised to the initial fluorescence (F/F0).
Axiovert confocal microscope
Manufactured by Zeiss
Sourced in United States
The Axiovert confocal microscope is a high-performance imaging instrument designed for advanced microscopy applications. It provides optical sectioning capabilities, allowing for the acquisition of detailed 3D images of samples. The core function of the Axiovert confocal microscope is to enable the study of complex biological and material samples with enhanced resolution and contrast.
Automatically generated - may contain errors
Lab products found in correlation
Lsm520 meta, by Zeiss (3 mentions)
Volocity software, by PerkinElmer (2 mentions)
Anti cleaved caspase 3 asp175, by Cell Signaling Technology (2 mentions)
Fluo 8 am, by Abcam (1 mentions)
Volocitytm software, by PerkinElmer (1 mentions)
Fluo 2am, by Thermo Fisher Scientific (1 mentions)
Fibronectin, by Merck Group (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Four chamber coverslips, by Thermo Fisher Scientific (1 mentions)
Stemspan sfem, by STEMCELL (1 mentions)
Imagem camera, by PerkinElmer (1 mentions)
Velocity software, by PerkinElmer (1 mentions)
Ec plan neofluar objective, by Zeiss (1 mentions)
Cy3 conjugated monoclonal donkey anti mouse igg secondary antibody, by Jackson ImmunoResearch (1 mentions)
Rabbit anti human aqp1, by Merck Group (1 mentions)
Anti phospho histone h3 ser10, by Cell Signaling Technology (1 mentions)
Cy3 conjugated monoclonal donkey anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Cy 3 conjugated monoclonal goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ab99302, by Abcam (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
12 protocols using axiovert confocal microscope
1
Islet Calcium Imaging using Fluo-8
2
Calcium Imaging of Pancreatic Islets
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Islets were incubated (37 °C 95% O2/5% CO2) for 45 min in 10 μm Fluo-2-AM (Invitrogen) dissolved in DMSO (0.01% w/v) and pluronic acid (0.001% w/v) in a bicarbonate buffer containing (in mm ) 120 NaCl, 4.8 KCl, 1.25 NaH2PO4, 24 NaHCO3, 2.5 CaCl2, 1.2 MgCl2, 3 d -glucose). Following incubation, islets were placed in a perifusion chamber, mounted on a Zeiss Axiovert confocal microscope, and perifused continuously at 34–36 °C with buffer containing the indicated glucose concentration. Fluo-2 was excited with a 491-nm laser, and emitted light was filtered at 525/50 nm. VolocityTM software (PerkinElmer) was used for data capture and analysis. Traces are presented as normalized intensity over time (F/Fmin).
3
Quantifying Hematopoietic Stem Cell Migration
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Four-chamber coverslips (Thermo Scientific) were coated with fibronectin (50 μg/ml in PBS; Millipore). Isolated HSPCs were plated at 250,000 cells/well on fibronectin in StemSpan SFEM (StemCell Technologies) media supplemented with murine cytokines IL-3, SCF, and FLT-3 (20 ng/ml; Peprotech) overnight at 37°C. For EHOP-016 treatment, isolated HSPCs were treated with 5 μM EHOP-016 for 1 h at 37°C prior to plating. Each well was washed twice with PBS to remove nonadherent cells. CO2-independent media supplemented with 0.5% fetal bovine serum (FBS) were added to each well for 1 h and migration was measured using a Zeiss Axiovert confocal microscope. Images were taken every 10 s for 50 min and were analyzed using Imaris tracking software (Bitplane Oxford Instruments) to measure track length, track displacement, and speed. The single cell–trajectory rose plots were produced from track position parameter data generated by Imaris tracking software. The beginning X, Y position and ending X, Y position of each HSPC were taken into account. Using the position data of each HSPC, each track was normalized to shift each HSPC to an origin of (0,0). A total of 15 positions between the beginning and end positions were randomly selected to produce a track. For each condition, a total of 10 HSPCs were assessed.
4
Intracellular Ca2+ Imaging in Pancreatic Islets
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Intracellular free Ca2+ was imaged in KHB saturated with 95% O2/5% CO2 and adjusted to pH 7.4, with additions as stated in the figures. Islets were incubated at 37°C for 45 minutes in KHB containing 10 μM Fluo-2-AM (Cambridge Biosciences). Islets were then transferred in a perfusion chamber, mounted on a Zeiss Axiovert confocal microscope, and continuously perfused at 34°C–36°C. Fluo-2 was excited with a 491-nm laser line and emitted light filtered at 525/50 nm. Images were acquired with a Hamamatsu ImagEM camera, and Volocity software (PerkinElmer) was used for data capture. Traces are presented as normalized intensity over time (F/F0) (49 (link)).
5
Functional Ca2+ Imaging of Pancreatic Islets
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Functional multicellular Ca2+-imaging was performed on isolated islets. Islets were incubated with Cal-520 (Stratech) for 45 min. at 37 °C in KREBS-Ringer buffer (KBH: 140 mM NaCl, 3.6 mM KCl, 0.5 mM NaH2PO4, 2 mM NaHCO3, 1.5 mM CaCl2, 0.5 mM MgSO4, 10 mM HEPES) supplemented with 3 mM glucose. Fluorescence imaging was performed on a Zeiss Axiovert confocal microscope using a Nipkow spinning disk head, and 10 × –20 × /0.3–0.5 NA, EC Plan-Neofluar objective (Zeiss) allowing rapid scanning of islet areas for long periods of time with minimal phototoxicity. Velocity software (PerkinElmer Life Sciences) provided the interface while islets were kept at 37 °C and constantly perifused with KHB containing 3 or 17 mM glucose or 20 mM KCl. For each experiment, 21–31 islets were used. Imaging data were analysed with ImageJ software using an in-house macro, available upon request.
6
Immunocytochemistry Procedure for Cleaved Caspase-3
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Immunocytochemistry was performed essentially as described in.45 Images were taken with a Zeiss Axiovert confocal microscope equipped with a LSM520 Meta. Anti-Cleaved Caspase-3 (Asp175) (Cell Signaling, cat no. 9661) was used at 1:400 dilution. Cy3-conjugated monoclonal donkey anti-mouse IgG secondary antibody was used at 1:400 (Jackson ImmunoResearch, cat. no.715–165–151).
7
Heat Shock-Induced Imaginal Disc Analysis
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48±4 hours old larvae of the desired genotype (see Fly stocks and crosses above) were subjected to heat shock at 37°C for 1 hour. The larvae were then placed at 25°C until L3 larvae had developed. The wing imaginal discs were then dissected in Ringer's solution and mounted on poly-l-lysine treated slides in Aqua-Poly/Mount. Images were taken with a Zeiss Axiovert confocal microscope equipped with a LSM520 Meta.
8
Immunofluorescence Staining of HCMV and AQP1
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The cytoslides were fixed in acetone for 5 min. Subsequently, a liquid repellent barrier was drawn using an Aqua-Hold Pap Pen, where urinary cells had been deposited by the cyto-centrifuge.
The mouse monoclonal anti-human cytomegalovirus immediate early antigen (HCMV-IEA) antibody (ARGEGE, Ref 11 − 003, France) was diluted 1:300 in PBS (phosphate buffered saline) with blocking solution (BSA, bovine serum albumin). The rabbit anti-human AQP1 was diluted 1:800 (Millipore AB 2219). Slide incubation for the primary antibody was carried out at 4˚C in a moist chamber and the next day with Alexa Fluor 488 goat anti-rabbit (diluted 1:400) and TRIC goat anti-mouse (diluted 1:400) for 1 h at room temperature. Five minutes before washing 40 µl of DAPI solution was added onto the slide for nuclear counterstain. After each antibody incubation, the slides were washed under constant stirring of the liquid in PBS. Finally, slides were mounted in Vectashield mounting medium for immunofluorescence (Vecotor Laboratories, Burlingham CA), covered with a coverslip and imaged with a Zeiss Axiovert confocal microscope and further processed by Adobe Photoshop version 6.
The mouse monoclonal anti-human cytomegalovirus immediate early antigen (HCMV-IEA) antibody (ARGEGE, Ref 11 − 003, France) was diluted 1:300 in PBS (phosphate buffered saline) with blocking solution (BSA, bovine serum albumin). The rabbit anti-human AQP1 was diluted 1:800 (Millipore AB 2219). Slide incubation for the primary antibody was carried out at 4˚C in a moist chamber and the next day with Alexa Fluor 488 goat anti-rabbit (diluted 1:400) and TRIC goat anti-mouse (diluted 1:400) for 1 h at room temperature. Five minutes before washing 40 µl of DAPI solution was added onto the slide for nuclear counterstain. After each antibody incubation, the slides were washed under constant stirring of the liquid in PBS. Finally, slides were mounted in Vectashield mounting medium for immunofluorescence (Vecotor Laboratories, Burlingham CA), covered with a coverslip and imaged with a Zeiss Axiovert confocal microscope and further processed by Adobe Photoshop version 6.
9
Immunocytochemistry and Confocal Imaging
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Immunocytochemistry was performed essentially as described in (Sullivan et al., 2000 ). Images were taken with a Zeiss Axiovert confocal microscope equipped with a LSM520 Meta. Primary antibodies used were anti-Pacman (Grima et al., 2008 (link)) (1:500), anti-Cleaved Caspase-3 (Asp175) (Cell Signaling, cat no. 9661;1:400), anti-phosphohistone H3 (Ser10) (Cell Signaling, cat. no. 9701; 1:400) and anti-Wingless (4D4) (Developmental Studies Hybridoma Bank; 1:400). Secondary antibodies used were Cy3-conjugated monoclonal goat anti-rabbit IgG (Jackson ImmunoResearch, cat. no. 711-165-152; 1:400), Cy3-conjugated monoclonal Donkey anti-mouse IgG (Jackson ImmunoResearch, cat. no.715-165-151; 1:400) and FITC-conjugated polyclonal goat anti-rabbit (Sigma, cat. no. F9887; 1:200).
10
Immunofluorescence Staining of TBX3
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Immunofluorescence was performed as previously described (16 (link)). Briefly, cells were incubated with rabbit polyclonal anti-TBX3 antibody (1:100 dilution; ab99302, Abcam, USA) at 4°C overnight. Subsequently, cells were incubated with fluor-conjugated secondary Cy3 donkey anti-rabbit IgG (1:1000 dilution; Jackson ImmunoResearch Laboratories, Inc., USA). To visualize nuclei, cells were incubated with 1 mg/mL Hoechst 33258 (Thermofisher Scientific, South Africa). Fluorescent cells were viewed using an Axiovert confocal microscope (Zeiss, USA).
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