Embryos were fixed for 20 min using 4% paraformaldehyde in PBS, devitelinized with a heptane:methanol 1∶1 mixture, and blocked with 0.1% Triton-X-100 in PBS (PBT) with 1% BSA for 15 min and later with 2% BSA in PBT for 30 min. Subsequently they were incubated with rabbit anti-GFP (1∶200 Torrey Pines Biolabs) antibody diluted in blocking solution containing 1% donkey serum for 2 h at room temperature. After washes embryos were incubated with an anti-rabbit-FITC (1∶200 Calbiochem) secondary antibody for 45 min. Muscleblind detection used sheep anti-Muscleblind (1∶500 [55] (link)) for 2 h followed by washes and primary antibody recognition with sheep biotin-conjugated secondary antibody (1∶100, Sigma) for 2 h. Then, washed embryos were incubated with ABC solution (ABC kit, VECTASTAIN) for 30 min at room temperature, and were washed and incubated with streptavidin-Texas Red (1∶1000, Vector) for 45 min. In all cases embryos were washed 3× with 1% BSA in PBT and were mounted in Vectashield (Vector) with 2 μg/ml DAPI. Images were taken on an Olympus FluoView FV100 confocal microscope. At least 10–15 embryos of the desired stage, and showing the relevant expression patterns, were analyzed.
Fluoview fv100 confocal microscope
Manufactured by Olympus
The FluoView FV100 is a confocal microscope designed for high-resolution imaging of fluorescent samples. It features a scanning laser that illuminates the specimen, and a pinhole that rejects out-of-focus light, enabling the capture of sharp, detailed images. The FluoView FV100 is capable of producing optical sections and 3D reconstructions of samples.
Automatically generated - may contain errors
Lab products found in correlation
Vectashield, by Vector Laboratories (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Texas red streptavidin, by Vector Laboratories (1 mentions)
Abc kit, by Vector Laboratories (1 mentions)
Lsm 780, by Olympus (1 mentions)
Fluoview viewer software, by Olympus (1 mentions)
Plan apochromat objective, by Olympus (1 mentions)
Bodipy, by Thermo Fisher Scientific (1 mentions)
Anti mouse immunoglobulin g igg, by Merck Group (1 mentions)
24 well plate, by BD (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
4 protocols using fluoview fv100 confocal microscope
1
Immunofluorescence Assay for Embryos
2
Immunofluorescence Imaging of 2D and 3D Cultures
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Cells were seeded on slides (2D culture) or on IBIDI glass bottom dishes (3D culture) and were cultured until desired confluency. Cells were fixed with 4% PFA in PBS+/+ (PBS with 0.9 mM CaCl2 and 0.5 mM MgCl2) for 15 min at room temperature for slide and 30 min for IBIDI. Immunofluorescence staining was performed as previously described 7. Confocal images were acquired with the Zeiss LSM 780 laser scanning confocal microscope using 40x Plan‐Apochromat objective (NA = 1.4) or with Olympus FluoView FV100 confocal microscope using 20x and 40x objective (NA = 0.75). Image acquisition software was ZEN (black edition, LSM 780; blue edition Cell Observer) and with Olympus FluoView viewer software, respectively.
3
Immunostaining of Drosophila Larvae
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Immunostaining of Drosophila larvae was carried out as described previously [31 (link)]. Briefly, third-instar (L3) larvae were dissected in chilled in Calcium-free HL3 solution [79 (link)], fixed in 4% formaldehyde in phosphate-buffered saline (PBS) for 30 min, permeabilised in 0.1% Triton X-100 in PBS and incubated with the appropriate antibodies. LDs were stained with 4,4-Difluoro-1,3,5,7,8-Pentamethyl-4-Bora-3a,4a-Diaza-s-Indacene 493/503 (BODIPY™, Thermo Fisher Scientific, Cat. No. D3922). Fixed and stained preparations were mounted in Vectashield (Vector Laboratories) and viewed using an Olympus FluoView FV100 confocal microscope. Images were acquired using a 60 × /1.5 NA objective and using FV10-ASX version 04.01 software. Unless otherwise stated, axonal analyses were conducted by imaging motor axon bundles passing through segment A7 [Additional file 2 : Supplementary Fig. 1, online resource]. Similarly, analyses of neuromuscular junctions (NMJs) were conducted by imaging muscles 6 and 7 at segment A7.
4
Analyzing MBNL1 Expression in DM1 Myotubes
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Immortalized DM1 fibroblasts were seeded at 4.0 × 104 cells per well in a 24-well plate (Falcon). After transfection and 4 days of differentiation, the transdifferentiated myotubes were fixed with 4% paraformaldehyde (PFA) for 15 min at room temperature (RT) and washed three times with 1× PBS. Myotubes were permeabilized with PBS-T (0.3% Triton-X in PBS) and blocked (PBS-T, 0.5% BSA, and 1% donkey serum) for 30 min at RT. They were then incubated with MBNL1 primary antibody (1:200, MB1a [4A8], Developmental Studies Hybridoma Bank52 (link)) at 4°C overnight. After three PBS-T washes, the cells were incubated for 1 h with a biotinylated anti-mouse-immunoglobulin G (IgG) (1:200, Sigma-Aldrich) and subsequent Avidin-Biotin amplification (Elite ABC kit, VECTASTAIN) for 30 min at RT. They were followed by three PBS-T washes and incubation with streptavidin-fluorescein isothiocyanate (FITC) fluorophore (1:200, Vector) for 2 h at RT. After three washes with PBS, the cells were mounted with VECTASHIELD mounting medium containing 2 μg/mL DAPI (Vector) to detect the nuclei.
Images of DM1 cells were taken on an Olympus FluoView FV100 confocal microscope. The images were taken at a 40× magnification and quantified using ImageJ with the following formula at a threshold of 10: mean pixel intensity = gray value/area.
Images of DM1 cells were taken on an Olympus FluoView FV100 confocal microscope. The images were taken at a 40× magnification and quantified using ImageJ with the following formula at a threshold of 10: mean pixel intensity = gray value/area.
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