RNA Detection

between about minutes 16 and 21. Collect eluate from instru- ment during these times. At minute 26, increase Buffer B to 95% an ...

Cap vial and place onto an orbital shaker set to about 200 rpm. Allow to shake for 20–25 min. Remove vial from shaker. Using a ...

the tube many times and continue until “Schlering lines” are no longer seen (seeNote 14). Place embryos at 37C for at least 1. ...

When embryos are in desired orientation, allow to dry for a few minutes at room temperature. This minimizes embryos shifting wh ...

tilting the glass down onto the embryos. This helps reduce the introduction of air bubbles. Use a paper towel to remove excess ...

specimens such as embryos, confocal microscopy is essential to visualize diffraction-limited objects. Wide-field fluorescence mi ...

Scanner settings and image acquisition: To improve our ability to locate and measure the signal of diffraction-limited objects, ...

maximize utilization of unique sequences, implement repeat masking. CG content of each oligonucleotide should be between 40 and ...

500 mL beaker with a stir bar and add 10 w/v % deionizing resin, e.g., mixed bed resin for deionizing (Sigma-Aldrich M8032). Sti ...

will begin mixing embryos and hybridization buffer while also splashing the sides of the tube in hybridization buffer. As mixing ...

nl/NyquistCalculator. This provides an initial estimate for ade- quate sampling density, that is, the number of pixels per unit ...

saturates the signal from transcription sites but provides high signal from individual mRNAs so that they may be separated from ...

Chapter 9 Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos L. Carine Stapel ...

use of up to 48 individually labeled 20 nucleotide-long oligonu- cleotides that hybridize to their target RNA [7]. The accumulat ...

2 Materials 2.1 Embryo Embedding Zebrafish embryos. Embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 , 0.33 mM MgSO 4 in d ...

PBS: 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.47 mM KH2PO4 in nuclease-free water. Adjust pH to 7.4 with HCl. PBT: 0.1 v/ ...

Glass pipet. 6-well staining plate (Fig.2a). Embryo manipulator (e.g., nickel-plated pin holder with curved pin, Fine Science T ...

Custom Stellaris smFISH probes, diluted to 25μM in Milli-Q water (seeNote 4). 1 mg/mL DAPI. Phalloidin–Alexa 488 (seeNote 5). G ...

(a) In the KNIME.ini file in your KNIME installation folder: set Xmx to –Xmx4g or –Xmx6g and –XX:MaxPermSize to –XX:MaxPermSize¼ ...

Embed multiple embryos in the cap of an Eppendorf tube (see Note 14) (Fig.2b). Fill the cap with OCT so that a dome of OCT exte ...