After PET/MR scanning had been completed, tissue containing sciatic nerve and adjacent muscle was rapidly harvested from both hind limbs of rats from each group. For whole nerve autoradiography, the nerves were exposed to a phosphor screen (medium MultiSensitive Phosphor Screen; PerkinElmer) for 12 h. The screen was imaged using a Typhoon 9410 Variable Mode Imager (Amersham Biosciences), and resulting images were analyzed by ImageJ (Image Processing and Analysis in Java, version 1.46; http://imagej.nih.gov/ij/index.html ). For nerve/muscle sections, tissue blocks were quickly frozen in optimal cutting temperature (O.C.T.) compound (Tissue-Tek, Sakura, USA) and 6 μm-thick sections were cut using a cryostat microtome HM500 (Microm) and mounted on microscope slides (Fisherbrand Superfrost® Plus Microscope Slides). The mounted sections were air-dried for 10 min and then exposed to 18F-sensitive storage phosphor screens (Perkin Elmer) for 12 h. The image plates were scanned with a Typhoon 9410 Variable Mode Imager (Amersham Biosciences), and resulting images were analyzed by ImageJ.
Typhoon 9410 variable mode imager
Manufactured by Cytiva
Sourced in United States
The Typhoon 9410 variable mode imager is a versatile laboratory instrument designed for high-sensitivity detection and quantification of biomolecules in a variety of applications. It features multiple excitation lasers and detection modes to accommodate a wide range of fluorescent and luminescent samples.
Automatically generated - may contain errors
Lab products found in correlation
Superfrost plus microscope slides, by PerkinElmer (3 mentions)
Cresyl violet acetate, by Merck Group (3 mentions)
Coomassie brilliant blue r 250, by Bio-Rad (2 mentions)
Superfrost plus microscope slide, by Thermo Fisher Scientific (1 mentions)
Bolt 8 bis tris plus gel, by Thermo Fisher Scientific (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Gel doc ez system, by Bio-Rad (1 mentions)
Mini protean tgx gel, by Bio-Rad (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
Readystrip ipg strip, by Bio-Rad (1 mentions)
Storage phosphor screen, by GE Healthcare (1 mentions)
Tnt quick coupled transcription translation system, by Promega (1 mentions)
Mini protean apparatus, by Bio-Rad (1 mentions)
Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions)
Lowry assay, by Bio-Rad (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
N glycosidase f, by New England Biolabs (1 mentions)
12 protocols using typhoon 9410 variable mode imager
1
Autoradiographic Analysis of Sciatic Nerve
2
Fluorescein-based Protein Binding Assay
3
Ex vivo Autoradiographic Analysis of Brain Tumors
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Example 10
Ex vivo Autoradiography: Autoradiography was performed as described in James et al., ((2012) J. Med. Chem. 55: 8272-8282, incorporated herein by reference in its entirety). In brief, coronal brain sections of U87 and GBM39 tumor-bearing mice were obtained 20 min after i.v. injection of 26 MBq [11C]DASA-23. Anesthetized mice were perfused with saline (10 mL) to remove intravascular [11C]DASA-23, and after cervical dislocation, the brain was removed and embedded in optimal cutting temperature (OCT) compound (Tissue-Tek) before being frozen on dry ice. Subsequently, 10 μm thick coronal brain sections were cut with a cryostat microtome HM500 (Microm). The sections were mounted on microscope slides (Fisherbrand Superfrost Plus microscope slides), air-dried for a minimum of 5 min, and then exposed to MultiSensitive storage phosphor screens (Perkin-Elmer) for 3 hours at −20° C. The image plates were analyzed with a Typhoon 9410 variable mode imager (Amersham Biosciences), and image data were visualized and processed by Image J.
4
PROTAC 2 Interaction with C-TDP-43 Aggregates
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Neuro-2a cells were seeded in 10 cm dishes at a concentration of 1 × 106 (blank control) or 2 × 106 (mCherry-C-TDP-43 overexpression) cells/mL. After overnight incubation, Neuro-2a cells were transfected with the mCherry-C-TDP-43 plasmid (10 μg) using Lipofectamine® 3000 (Invitrogen) and incubated for another 24 h. The transfected cells were then harvested with RIPA buffer containing complete protease inhibitor (Roche) and sonicated on ice (10 s, two repetitions). Next, PROTAC 2 at various concentrations (0, 5, 10, 20, and 40 μM) was added to each extract (identical protein quantity, 100 μg). The mixtures (PROTAC 2 + cell lysate) were gently shaken at 4 °C for 2 h. After that, the mixtures were fractionated and the RIPA-insoluble fraction were resuspended with RIPA buffer and loaded on CA membrane by applying filter trap assay. The fluorescent PROTAC 2 retained on C-TDP-43 aggregates was detected by Typhoon9410 Variable Mode Imager (Amersham BioScience, Piscataway, NJ, USA) (λex = 457 nm, λem = 488 nm).
5
VSV-EGFP Plaque Reduction Assay in Huh-7.5 Cells
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The plaque reduction assay using the IFN-sensitive VSV-EGFP23 (link) for the infection of Huh-7.5 cells was performed following an earlier described method22 (link). Huh-7.5 cells (5 × 105/well) seeded in 12-well plates were treated with the test compound for 24 h. Cells were then washed with PBS twice and inoculated with VSV-EGFP (200 plaque forming units [PFU]/well). After 1 h of infection, the cells were washed again with PBS and overlaid with media containing 1% methylcellulose. Fluorescent viral plaques were subsequently scanned and quantitated at 24 h postinfection using the Typhoon 9410 variable mode imager (Amersham Biosciences; Baie d’Urfe, Quebec, Canada). Data were calculated as percent inhibition against VSV-EGFP infection relative to the DMSO treatment control.
6
In Vivo Imaging of Neuroinflammation
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Mice were administered LPS (5 mg/kg)
or saline (100 μL) intraperitoneally. Approximately 24 h later,
the11 C-MGX-10S radiotracer (800–1200
μCi) was administered intravenously; mice utilized in the blocking
study were treated with GLPG1205 (1 mg/kg intravenous) 10 min prior
to radiotracer administration. 20 minutes after radiotracer injection,
each mouse was perfused with saline (20 mL) and brains were removed
and snap-frozen in dry ice and then embedded in the optimal cutting
temperature compound (Tissue-Tek, Sakura, U.S.). Next, brains were
sectioned sagittally (60 μm) using a cryostat microtome HM500
(Microm, Germany). The sections were mounted on microscope slides
(Fisherbrand Superfrost Plus microscope slides), air-dried for 10
min, and then exposed to phosphor imaging film (PerkinElmer, U.S.)
for 12 h at 4 °C. The image plates were analyzed using a Typhoon
9410 variable mode imager (Amersham Biosciences, U.S.), and image
data were visualized and processed by ImageJ (National Institute of
Health). Anatomy of brain sections was confirmed by Nissl staining
(cresyl violet acetate, Sigma-Aldrich).
or saline (100 μL) intraperitoneally. Approximately 24 h later,
the
μCi) was administered intravenously; mice utilized in the blocking
study were treated with GLPG1205 (1 mg/kg intravenous) 10 min prior
to radiotracer administration. 20 minutes after radiotracer injection,
each mouse was perfused with saline (20 mL) and brains were removed
and snap-frozen in dry ice and then embedded in the optimal cutting
temperature compound (Tissue-Tek, Sakura, U.S.). Next, brains were
sectioned sagittally (60 μm) using a cryostat microtome HM500
(Microm, Germany). The sections were mounted on microscope slides
(Fisherbrand Superfrost Plus microscope slides), air-dried for 10
min, and then exposed to phosphor imaging film (PerkinElmer, U.S.)
for 12 h at 4 °C. The image plates were analyzed using a Typhoon
9410 variable mode imager (Amersham Biosciences, U.S.), and image
data were visualized and processed by ImageJ (National Institute of
Health). Anatomy of brain sections was confirmed by Nissl staining
(cresyl violet acetate, Sigma-Aldrich).
7
Brain Proteome Analysis by 2DE
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Two dimensional electrophoresis (2DE) of the brain proteins was performed using a previously published method (Wu et al., 2012 (link); Zhang et al., 2013 (link)). Briefly, the commercial 24-cm long ReadyStrip IPG strips (pH 5–8, linear, Bio-Rad) in PROTEAN IEF (isoelectric focusing) Cell (Bio-Rad) were used for the first dimension separation. Each strip was loaded with 250 μg protein in total. After the IEF is completed, the strips were equilibrated with an IEF buffer containing 1% dithiothreitol (DTT) and 2.5% iodoacetamide for 15 min. Subsequently, the strips were run on the 11% SDS-PAGE gel at 80 V for 40 min, followed by 200 V until the second dimension separation finished. All gels were silver-dyed with an improved mass spectroscopy-compatible method and scanned at a resolution of 500 dpi on a Typhoon 9,410 Variable Mode Imager (Amersham Biosciences).
8
Ex Vivo Autoradiography of Brain Sections
9
Mitochondrial Protein Import Assay
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[35S]‐methionine‐labeled proteins were generated with the TNT Quick Coupled Transcription/Translation System (Promega) according to the manufacturer's instructions. Labeled proteins were incubated with isolated mitochondria prepared by differential centrifugation from control and patient fibroblasts grown in glucose (Reyes et al, 2011 ) and import were carried out at 37°C for 1–30 min. A negative control containing 1 μM FCCP was included for the longest time point. Mitochondria from TFAM import reactions were washed three times before being subjected to SDS–PAGE, while in AAC1 import experiments, mitochondria were trypsin treated for 15 min at 37°C before the washes and SDS–PAGE. Gels were dried, exposed to storage phosphor screens (GE Healthcare), visualized on the Typhoon 9410 Variable Mode Imager (Amersham Biosciences), and quantified using ImageJ.
10
Ex vivo Autoradiography of 64Cu-TIMP2 in Mouse Brain
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