The normal Balb/c mouse (male, 10–12 weeks-old) frozen brain sections (10-μm thickness) were prepared using a cryostat microtome (CM1900, Leica, Germany) one day before and kept in −80℃ freezer. The sections were first thawed at room temperature for 20 min and rehydrated with ice-cold PBS buffer (pH 7.4) for 5 min. Then all sections were incubated with 4 nM [18F]9e with, or without, 10 μM olaparib at room temperature for 1 h to define the control and nonspecific binding groups. After the radioligand incubation, all of them were washed three times with ice-cold PBS for 3 min, dried up with a fan, and exposed to a BAS-SR 2040 imaging plate (20×40 cm, Fujifilm, Japan) for 2 hr. In the end the plate were scanned with a Typhoon 7000 phosphorimager (GE Healthcare) in a condition of 500 V and a resolution of 25 μm.
Typhoon 7000 phosphorimager
Manufactured by GE Healthcare
The Typhoon 7000 phosphorimager is a lab equipment designed for the detection and analysis of radioactively labeled molecules, such as proteins and nucleic acids, in gel-based experiments. It uses a sensitive photomultiplier tube to detect and digitize the radioactive signal, providing high-resolution images for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (4 mentions)
Oligo dt primer, by Promega (2 mentions)
Goscript reverse transcription system, by Promega (2 mentions)
Gotaq green, by Promega (2 mentions)
Bas sr 2040 imaging plate, by Fujifilm (1 mentions)
Cm1900, by Leica (1 mentions)
Ecl plus reagent, by GE Healthcare (1 mentions)
Phosstop, by Roche (1 mentions)
Cryomicrotome, by Thermo Fisher Scientific (1 mentions)
Imagequant tl version 8, by GE Healthcare (1 mentions)
6 protocols using typhoon 7000 phosphorimager
1
Autoradiographic Quantification of PARP Inhibitor Binding
2
Immunoblotting Quantification of FOXM1 and G6PD
3
Splicing Analysis of CFTR Exons
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RNA was extracted from cells using TRIzol according to manufacturer instructions (Thermo Fisher Scientific). RNA was extracted from differentiated cells grown on filters by cutting out the filter and placing directly in TRIzol. Reverse transcription was performed on total RNA using the GoScript Reverse Transcription System with an oligo-dT primer (Promega). Splicing was analyzed by radiolabeled PCR of resulting cDNA using GoTaq Green (Promega) supplemented with α-32P-deoxycytidine triphosphate (dCTP). Primers for amplification are reported in Supplementary Table S1 and include primer sets flanking human CFTR exons 22 and 24 (hCFTRex22F, hCFTRex24R), and primers for human GAPDH, human β-actin (hβ-actinFor, hβ-actinRev) and SRSF2 1.7 kb (hSC351.7kbF, hSC351.7kbR) (34 (link)). Reaction products were run on a 6% non-denaturing polyacrylamide gel and quantified using a Typhoon 7000 phosphorimager (GE Healthcare) and ImageJ software (35 (link)).
4
Cryosectioning and Phosphor Imaging
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Dissected tumors were embedded in an optimal-cutting-temperature compound (Tissue-Tek; Sakura) and immediately snap-frozen on dry ice. The specimens were sectioned to a thickness of 6 μm using a cryomicrotome (Thermo Fisher Scientific) and mounted on slides. The slides were exposed to a photostimulable phosphor plate for 24 h and read using a Typhoon7000 phosphor imager (GE Healthcare Life Sciences). The recorded images were analyzed using ImageQuant TL, version 8.1 (GE Healthcare Life Sciences).
5
Analyzing Splicing by Radiolabeled PCR
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RNA was extracted from cells using TRIzol, according to manufacturer instructions (Thermo Fisher Scientific). Reverse transcription was performed on total RNA using the GoScript Reverse Transcription System with an oligo-dT primer (Promega). Splicing was analyzed by radiolabeled PCR of resulting complementary DNA (cDNA) using GoTaq Green (Promega) spiked with α-32P-deoxycytidine triphosphate. Primers for amplification are reported in SI Appendix, Table S1 . Reaction products were run on a 6% nondenaturing polyacrylamide gel and quantified using a Typhoon 7000 phosphorimager (GE Healthcare) and ImageJ software (74 (link)).
6
MAPK Pathway Phosphorylation Profiling
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