Drosophila neuromuscular junction ultrastructure was imaged following standard Electron Microscopy procedures. Briefly, wandering third instar larvae were filleted and dissected at room temperature in 2.5 mM calcium HL-3 medium and subsequently fixed overnight in 2% paraformaldehyde/2.5% glutaraldehyde/0.1 M cacodylic acid (pH 7.2). The fixed fillets were then processed inside a Ted Pella Bio Wave microwave with the vacuum attachment. Samples were fixed again, followed by 3× water rinses, post-fixed with 1% aqueous osmium tetroxide, and followed again with 3 more rinses with Millipore water. A graded series of ethanol concentrations from 30–100% was used as the initial dehydrant followed with propylene oxide as a final dehydrant. Samples were gradually infiltrated with 3 propylene oxide and Embed 812 graded ratios into 3 changes of pure resin under vacuum. Samples were allowed to infiltrate in pure resin overnight on a rotator. The samples were embedded into flat silicone moulds and cured in the oven at 62°C for three days. The polymerized samples were sectioned and stained with 1% uranyl acetate for ten minutes followed by lead citrate for one minute before TEM examination. TEM images were captured using a JEOL JEM 1010 transmission electron microscope with an AMT XR-16 mid-mount 16 mega-pixel digital camera.
Bio wave microwave
Manufactured by Ted Pella
The Bio Wave is a microwave oven designed for laboratory use. It provides a controlled heating environment for various applications in the scientific field.
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Lab products found in correlation
3 protocols using bio wave microwave
1
Drosophila Neuromuscular Junction Ultrastructure
2
Microwave-Assisted SEM Preparation
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Sorted trichomonads or cecum from mono-colonized mice were prepared for scanning electron microscopy (SEM) via a Biowave microwave-assisted (Ted Pella, Inc.) protocol as described in (Jackson et al. 2018 (link)). Briefly, samples were fixed in 2% paraformaldehyde, 2.5% glutaraldehyde, 0.05% alcian blue in 0.1 M Sorenson’s phosphate buffer (Karnovsky’s Fixative, Electron Microscopy Sciences) and stored at 4 °C until further processed. Sorted cells were adhered to poly-L-lysine coated silicon wafer chips for 30 minutes. Both sorted cells and cecum pieces were post-fixed in 0.5% OsO4, 0.8% K4Fe(CN)6 in 0.1 M sodium cacodylate buffer for 1 hour. The samples were rinsed in dH20, stained with 1% aqueous tannic acid for 1 hour, rinsed with dH20, and finally fixed with a second round of 0.5% OsO4, 0.8% K4Fe(CN)6 in 0.1 M sodium cacodylate buffer for 1 hour to enhance conductivity under the scanning electron beam. Samples were rinsed with dH20, then dehydrated using a graduated ethanol series into 100% ethanol, critical point dried using a Bal-Tec CPD 030 (Leica Microsystems, Buffalo Grove, IL), placed on a stub using carbon sticky tape, and sputter coated with 10Å of iridium (EMS300TD, Electron Microscopy Sciences). Samples were placed in a Hitachi SU8000 scanning electron microscope operating at 5.0 kV, 10 μA, and a working distance of ~8 mm using a secondary electron detector.
3
Formaldehyde-Glutaraldehyde Fixation and DAPI Staining
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Cells were fixed with a mixture of 4% formaldehyde and 0.5% glutaraldehyde (EM grade EMS) in 0.1 M PHEM Buffer (pH 6.9: 240 mM PIPES [Sigma-Aldrich], 100 mM Hepes [Biomol], 8 mM MgCl2 [Merck], and 40 mM EGTA [Sigma-Aldrich]). A Ted Pella BioWave microwave with a temperature control unit (Pelco BioWave microwave with ColdSpot [Ted Pella, Inc.]) was used to accelerate the fixation process to 14 min at 250 W. DAPI (1 µg/ml in 0.1 M PHEM; Thermo Fisher Scientific) was applied to cells to stain the nucleus for a total of 10 min. To quench glutaraldehyde auto-fluorescence, cells were rinsed with 150 mM glycine (Merck) in PHEM buffer. Cells were left in the PHEM buffer for imaging.
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