Perfusion of mice and immunostaining for myelin proteins were performed as described (Trapp et al. 1997 (link)) with slight modifications. Immunohistochemistry for phosphorylated antibodies was done using the protocol of Narayanan et al. (2009) (link), with some modifications. Phospho-S6 ribosomal protein antibody was used to test section quality for all samples. Only sections with robust and uniform phospho-S6 ribosomal protein signal in the neocortex were chosen for further staining with other antibodies. On day 1, brains were sectioned at 30 μm on a sliding microtome. Free-floating sections were washed three times in PBS and antigen retrieval was performed with 10 mM sodium citrate (pH6.0) at 65°C for 10 min in a BioWave (Ted Pella). Sections were rinsed in PBS twice, blocked in blocking solution (200 mM NaCl, 50 mM Tris. HCl, 100 mM L-lysine, 150 mM glycine) with 5% normal donkey serum (NDS) and 3% Triton X-100 for 1 h at RT and incubated in primary antibodies in 3% NDS containing 1% Triton X-100 for 72 h at 4°C. All phospho-antibodies and Sox10 were used at 1:200. On day 4, sections were washed three times in PBS, incubated with secondary antibodies (1:800) at RT, then counter stained for DAPI for 5 min. Finally, sections were washed three times in PBS and mounted in Vectashield (Vector Laboratories).
Bio wave
Manufactured by Ted Pella
Sourced in United States
The Bio Wave is a laboratory equipment designed for cell culture applications. It provides a controlled environment for the growth and maintenance of cells. The device is capable of regulating temperature, humidity, and gas composition to ensure optimal conditions for cell culture.
Automatically generated - may contain errors
Lab products found in correlation
Embed 812 resin, by Electron Microscopy Sciences (4 mentions)
Formvar coated copper grid, by Electron Microscopy Sciences (3 mentions)
Jem 1010, by JEOL (3 mentions)
Uc7 microtome, by Leica camera (2 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Temperature control unit, by Ted Pella (1 mentions)
Uc7 microtome, by Electron Microscopy Sciences (1 mentions)
Alexa fluor 488 conjugated anti rabbit, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 conjugated anti mouse, by Thermo Fisher Scientific (1 mentions)
Pronase, by Roche (1 mentions)
Hyaluronidase, by Roche (1 mentions)
Coldspot, by Ted Pella (1 mentions)
8 protocols using bio wave
1
Immunostaining and Quantification of Myelin Proteins
2
Microwave-Assisted Preparation of Photonic Chip Samples for SEM
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All processing was done in a Ted Pella Biowave with a temperature control unit (Ted Pella Inc.)5 (link),40 . Because the photonic-chips were overheating upon microwave processing, the samples were placed directly on the control unit, set to 4 °C, and the vacuum chamber was inverted on top. To stabilize the cells further after light microscopy the samples were processed for 14 min (2 min vacuum on-off-on-off-on-off-on, 100 W) in fixative and washed twice with 0.1 M cacodylate buffer. Post-fixation was done with 1% Osmium tetroxide, 1% K3Fe(CN)6 in 0.1 M cacodylate. The cells were post-stained with 1% tannic acid and 1% uranyl acetate. Samples were then dehydrated in increasing ethanol series (30%, 50%, 75%, 90% and 2 × 100%) and embedded in increasing amounts of Durcupan (30%, 50%, 75%, 90%). To be able to remove as much resin as possible, the resin exchange steps were increased to 90% Durcupan in EtOH and not 100% Durcupan. The chips were centrifuged for 30 min at 37 °C in a vertical position to further remove excess resin, and polymerized in the oven for 96 h at 60 °C. The chips were then cut to a final size of 1 cm2 to fit the SEM stubs.
3
Transmission Electron Microscopy of Adult Fly Laminae
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Laminae in adult flies were processed for TEM imaging as described [52 (link)]. Samples were processed using a Ted Pella Bio-Wave microwave oven with vacuum attachment. Adult fly heads were dissected at 25°C in 4% paraformaldehyde, 2% glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.2). Samples were subsequently fixed at 4°C for 48 hours. 1% osmium tetroxide was used for secondary fixation and subsequently dehydrated in ethanol and propylene oxide, and then embedded in Embed-812 resin (Electron Microscopy Science, Hatfield, PA). 50 nm ultra-thin sections were obtained with a Leica UC7 microtome and collected on Formvar-coated copper grids (Electron Microscopy Science, Hatfield, PA). Specimens were stained with 1% uranyl acetate and 2.5% lead citrate and imaged using a JEOL JEM 1010 transmission electron microscope with an AMT XR-16 mid-mount 16 megapixel CCD camera.
4
Transmission Electron Microscopy of Adult Fly Retinas
5
Drosophila Retina Ultrastructure Imaging
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Drosophila retina ultrastructure was imaged following standard electron microscopy procedures using a Ted Pella Bio Wave processing microwave with vacuum attachments. Briefly, fly heads were dissected and fixed at 4°C in fixative (2% paraformaldehyde, 2.5% glutaraldehyde, 0.1 M sodium cacodylate, and 0.005% CaCl2, pH 7.2) overnight. The samples were then postfixed in 1% OsO4, dehydrated in ethanol and propylene oxide, and then embedded in Embed-812 resin (Electron Microscopy Sciences) under vacuum. Photoreceptors were then sectioned and stained in 1% uranyl acetate and saturated lead nitrate. TEM images of photoreceptor sections were taken using a JEOL JEM 1010 transmission electron microscope at 80 kV with an AMT XR-16 mid-mount 16 mega-pixel digital camera.
6
TEM Imaging of Drosophila Retinae and Laminae
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Both retinae and laminae in adult flies were processed for TEM imaging as described [58 (link)]. Samples were processed using a Ted Pella Bio Wave microwave oven with vacuum attachment. Adult fly heads were dissected at 25 °C in 4 % paraformaldehyde, 2 % glutaraldehyde, and 0.1 M sodium cacodylate (pH 7.2). Samples were subsequently fixed at 4 °C for 48 hours. 1 % osmium tetroxide was used for secondary fixation and subsequently dehydrated in ethanol and propylene oxide, and then embedded in Embed-812 resin (Electron Microscopy Science, Hatfield, PA). 50 nm ultra-thin sections were obtained with a Leica UC7 microtome and collected on Formvar-coated copper grids (Electron Microscopy Science, Hatfield, PA). Specimens were stained with 1 % uranyl acetate and 2.5 % lead citrate and imaged using a JEOL JEM 1010 transmission electron microscope with an AMT XR-16 mid-mount 16 mega-pixel CCD camera. For quantification of ultrastructural features, electron micrographs were examined from 3 different animals per genotype.
7
Immunofluorescence Imaging of Alzheimer's Disease
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All paraffin-wax-embedded sections of pellet and native AD tissues were pretreated for antigen retrieval using citrate buffer (pH 6) in a BioWave ® equipped with the ColdSpot ® system (Ted Pella Inc., Redding, CA, USA), as previously described (Anderson et al., 2018) . Then, sections were treated with 1 mg/mL pronase (Roche Diagnostics Corp.) in PBS for 30 min at room temperature followed by 1 mg/mL hyaluronidase in PBS for 30 min at 37 °C. Blocking buffer composed of 5 % BSA was applied for 45 min and, then, sections were probed with the primary antibodies (Table 2). Primary antibodies were diluted in 1 % BSA in PBS, then left on tissue sections overnight at 4 °C. Alexa Fluor 594-conjugated anti-mouse (1:250; Life Technologies) and Alexa Fluor 488-conjugated anti-rabbit (1:200; Life Technologies) secondary antibodies were diluted in 1 % BSA and incubated on the sections for 45 min at room temperature. For all tissues prepared as negative controls, all steps were performed similarly except for modification to the antibody step. Three experimental negative controls were used, including (1) PBS replacing the primary antibody, (2) tissues incubated with a non-immune antibody of the same species/ isotype/concentration, and (3) secondary antibody replaced with PBS. Slides were mounted using ProLong Gold anti-fade reagent containing DAPI
8
Electron Microscopy Sample Preparation
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A PELCO Biowave equipped with a ColdSpot (Ted Pella) was used for all processing steps except where stated otherwise. For the GA/FA fixation and OsO4 post-fixation the microwave was operated at 100W cycling intervals of 2 min under vacuum at room temperature. For Thiocarbohydrazide ((NH 2 NH) 2 CS) the same settings were used at 40 C and for UA and lead aspartate (C 4 H 5 NO 4 Pb) at 50 C. Quenching, rinses and dehydration were 40 sec long at 250W with no vacuum at room temperature. The resin infiltration was a 5-step-graded series of EtOH/resin followed by 2 changes of 100% resin; these steps were 3 min long at 250W under vacuum at room temperature.
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