RNA Detection

Fig. 3Thresholding parameters for counting spots in the FindFoci ImageJ plugin. With these settings (a) the “Background param” s ...

3.9.2 FISHQuant (Fig.4c) 1. Save images to be analyzed into separate channels, FISH chan- nel and marker channels for segmentati ...

Export the thresholded spots text file to determine the number of transcripts. 3.9.3 Imaris Spots (Fig.4d) Open the image wit ...

dye of choice. The further the dye chosen emits into the red wavelengths, the better the signal–noise ratio because of reduced t ...

Trcek T et al (2015) Drosophila germ granules are structured and contain homotypic mRNA clusters. Nat Commun 6:7962 Karr J et a ...

Chapter 11 Detection of mRNA and Associated Molecules by ISH-IEM on Frozen Sections Catherine Rabouille Abstract The use of tagg ...

with specific antibodies that are detected by enzymatic reactions (alkaline phosphatase and peroxidase). So far, this technique ...

2% PFA + 0.2% glutaraldehyde (GA) in 0.1 M phosphate buffer at pH 7.4. Prepare fresh from 16% PFA and 8% EM grade GA stocks. 1% ...

Methylcellulose (MC): 25 centipoises. 2 w/v% MC stock solution was prepared and stored as described in [4]. Methylcellulose–suc ...

Rabbit anti-biotin (Rockland) to detect the biotinylated probe, diluted 1:10,000 in 1% BSA in PBS. To detect the DIG-labeled pr ...

Cut ultrathin frozen sections (70–80 nm) at 120 Cinan ultracryomicrotome [4] and collect them with a drop of methylcellulose– ...

Prehybridization: After rinsing the fixative on three drops of PBS at 37C, incubate the grids (sections side down) in pre- hyb ...

For protein detection after mRNA, proceed with double or triple labeling of the sections, by repeatingsteps 1–4using appropriat ...

as copper dissolves in the hybridization buffer. Sections might fold or be lost from the grids during the procedure and this can ...

References Lawrence JB, Singer RH (1986) Intracellular localization of messenger RNAs for cytoskele- tal proteins. Cell 45(3):4 ...

Chapter 12 Hybridization Chain Reaction for Direct mRNA Detection Without Nucleic Acid Purification Yao Xu and Zhi Zheng Abstrac ...

a(Toehold) b(Stem) c(Loop) b c b a b Hairpin set (^) Triggers chain reaction Cascade chain reaction ... Target (initiator)^ a b ...

sufficient for clinical applications. Linear, one-dimensional chain reactions are adopted in most current HCR amplification meth ...

Capture Probe CEs LEs Mix lysate with all probes Add hairpin Set X Add hairpin Set A Capture Stage Amplification Stage !!! Adapt ...

UV transilluminator. Resolving gel buffer and running buffer (1
TBE): 90 mM Tris, 89 mM boric acid, and 2.0 mM EDTA, pH 8.0. Ad ...