Whole flies, larvae, and fly eyes were imaged with Nikon SMZ1500 and Leica MZ16 F microscopes and deconvolved with Helicon Focus software (HeliconSoft). 3rd instar larvae eye inmaginal discs were dissected in PBS, fixed with 4% paraformaldehyde on PBS for 20 minutes at room temperature, washed with PBS, quenched with 1M glycine on PBS, washed again, and mounted with Vectashield media (Vector Laboratories). Confocal images were acquired with Nikon Ti Spinning Disc confocal microscope equipped with 20x objective and processed using Imaris software (Bitplane).
Vectashield media
Manufactured by Vector Laboratories
Sourced in United States
Vectashield is a mounting media designed for fluorescence microscopy. It is a water-based, glycerol-based solution that is optimized to preserve fluorescent signals in fixed samples. Vectashield helps to reduce photobleaching and maintain the integrity of fluorescent labels during imaging.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 780 confocal microscope, by Zeiss (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (5 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (3 mentions)
Cy3 conjugated, by Jackson ImmunoResearch (2 mentions)
Plan apochromat 20 na 0, by Zeiss (2 mentions)
Plan apochromat 40 na1, by Zeiss (2 mentions)
Phalloidin tritc, by Merck Group (2 mentions)
Alexa fluor tagged secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Donkey serum, by Jackson ImmunoResearch (2 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (2 mentions)
Donkey anti mouse cy3, by Jackson ImmunoResearch (2 mentions)
Donkey anti rat cy3, by Jackson ImmunoResearch (2 mentions)
Mouse anti flag, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Smz1500, by Leica camera (1 mentions)
Mz16f, by Leica camera (1 mentions)
Focus software, by Helicon (1 mentions)
Deltavision, by Cytiva (1 mentions)
Softworks, by Cytiva (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
21 protocols using vectashield media
1
Imaging Drosophila Larval Eye Discs
2
Immunofluorescence Imaging of Spermatocytes
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Cells were cultured on coverslips in the presence or absence of 100 nM camptothecin for 22 h. Cells were permeabilized with CSK buffer (10 mM PIPES at pH 6.8, 100 mM NaCl, 300 mM Sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 1× protease inhibitor cocktail, 1× phosphatase inhibitor) for 5 min on ice and fixed with 2% PFA (Sigma) for 15 min at room temperature. The coverslips were blocked in TBST containing 3% BSA. Subsequently, coverslips were incubated with primary antibodies in TBST containing 3% BSA for overnight, washed 3 times with TBST and secondary antibodies for 1 h. After washing with TBST, the coverslips were mounted with Vectashield media (Vector Laboratories).
Stained spreads were observed using an epi-fluorescence microscope (BX51; Olympus) with a 60 × objective (NA1.3). Images were captured by CCD camera (CoolSNAP; Roper). Mouse spermatocyte spreads were observed using a computer-assisted fluorescence microscope system (DeltaVision; Applied Precision). The objective lens was an oil immersion lens (100×; NA, 1.35). Image deconvolution was performed using an image workstation (SoftWorks; Applied Precision), and afterwards processed using iVision (Sillicon), and Photoshop (Adobe) software tools.
Stained spreads were observed using an epi-fluorescence microscope (BX51; Olympus) with a 60 × objective (NA1.3). Images were captured by CCD camera (CoolSNAP; Roper). Mouse spermatocyte spreads were observed using a computer-assisted fluorescence microscope system (DeltaVision; Applied Precision). The objective lens was an oil immersion lens (100×; NA, 1.35). Image deconvolution was performed using an image workstation (SoftWorks; Applied Precision), and afterwards processed using iVision (Sillicon), and Photoshop (Adobe) software tools.
3
Immunostaining of Drosophila Embryos and Ovaries
4
Confocal Microscopy of 5-ALA Skin Uptake
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For confocal microscopy assay, the mice were treated with either the control (PBS), 5-ALA, 5-ALA/DPPC (0.5% DPPC). These were the three groups for our experiment. After 4 hours, the skin where the drug was administrated was harvested and fixed with 4% paraformaldehyde. Then tissue samples were sliced into 5 μm sections by frozen section and mounted with Vectashield media (Vector Laboratories) for laser confocal microscopy observation (model FV1000, Olympus).
5
Immunohistochemical Analysis of Pancreatic Tissues
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Fixed human cadaver pancreatic tissue (Prodo) was incubated in 30% sucrose solution followed by embedding in OCT (TissueTek), cryopreservation, and sectioning. Dissected mouse pancreatic tissues were fixed with Z-fix (Anatech), incubated in 30% sucrose overnight, and cryopreserved. Tissue sections were stained with primary antibodies, anti-mouse vimentin (1 : 200, Sigma), anti-human vimentin (1 : 50, Calbiochem), anti-YFP/GFP (1 : 500, Abcam), anti-Galectin-1 (1 : 50, RnD Systems), anti-LAMA2 (1 : 200, Enzo), and anti-PDX1 (1 : 200, RnD Systems), followed by staining with AlexaFluor tagged secondary antibodies (1 : 500, Invitrogen) and mounting Vectashield media (Vector). Nuclei were visualized with DAPI. Images were acquired using a Leica SP5 microscope or an InCell Analyzer 2000 for quantification (GE Healthcare). 16 fields from each condition were randomly selected by the InCell Analyzer and imaged for quantification. PDX1 positive nuclei over total nuclei were determined using InCell Developer software (GE Healthcare).
6
Ligand-Induced β-Arrestin2 Recruitment Assay
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Ligand-induced recruitment of β-arrestin2YFP was assessed in GBR-24 cells, which are HEK293/glosensor (Gs22A)-derived cells stably expressing β-arrestin2YFP 42 . The cells in six-well plates were transiently transfected with plasmid DNA encoding PTH1R-WT or mutant derivative, and 24 hours later seeded onto glass coverslips in 6-well plates. At 48 hours post-transfection, the cells were treated with PTH(1-34)TMR, PTHrP(1-36)TMR or Ile5-PTHrP(1-36)TMR (10 or 30 nM) in HB for 30 min at room temperature; the cells were then rinsed with HB, fixed for 5 min in 3.7% paraformaldehyde buffer (Boston BioProducts Cat. No. BM-158), rinsed with HB, mounted on a glass microscope slide in Vectashield media containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Cat. No. H1500) and imaged at 400X magnification using a Nikon Eclipse fluorescence microscope equipped with a CCD camera configured with SPOT imaging software. In some experiments, the cells were imaged by confocal microscopy using a Leica DMi8 inverted Microscope at ×400 magnification, and the images were processed using Leica Application Suite X (LAS X) software. Regions of interest were digitally expanded 3-5X to improve views.
7
Immunohistochemical Localization of PDE6β
8
SYTOX Green Viability Assay
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Control and HPRT-deficient cells were seeded in the conditions indicated above and treated with vehicle PBS control and 100 µM of hydrogen for at least four hour, afterward the cells were washed twice with 1X PBS and exposed to 100 nM of SYTOX green (Life Technologies) in 1X PBS for 10 min. Subsequently, the SYTOX dye was removed and the cells washed twice with 1X PBS and fixed with 4% paraformaldhyde for 3 min and then washed with 1X PBS. The resulting cells were mounted in Vectashield media containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories). Fluorescence was visualized using Olympus BX51 fluorescent microscope with BP72 Olympus acquisition camera.
9
Quantitative Analysis of Embryonic Tissues
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Embryos were dissected in PBS and fixed for 20 min in 4% PFA. After fixation, immunostaining was performed as previously described (Baumgardt et al., 2014 (link)). Embryos were mounted in Vectashield media (Vector Laboratories). Semi-automated macro, ImageJ macro language-based, was designed and used for the quantification of the volume of each segment with the Fiji software. The same software was used to manually count the number of cells and volumes of areas of nerve cord in order to estimate average single cell volume (Yaghmaeian Salmani et al., 2018 (link)) (see Figure 2—source data 1 for details).
10
Immunostaining of Drosophila Embryos and Ovaries
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Hand-devitellinized embryos and dissected larval ovaries were immunostained as previously described34 (link),35 (link). Samples were mounted in Vectashield® media containing DAPI (Vector Laboratories). Primary antibodies: rabbit anti-Vasa serum (1:5000, R Lehmann) and mouse anti-1B1 (1:20, DSHB). Alexa Fluor 488- (Life Technologies) and Cy3-conjugated (Jackson Immunoresearch) secondary antibodies were used at a 1:500 dilution. Phalloidin-TRITC (Sigma) was used at 1:250 (from 20μM stock). Fluorescent images were acquired with Plan-Apochromat 20×/NA0.8 and Plan-Apochromat 40×/NA1.4 (oil immersion) objectives on a Zeiss LSM 780 confocal microscope.
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