A fragment of NK2.1, and the full-length sequences of foxQ2, FGF8/17/18, GATA456, HNF4, wnt1, evx, and twi [GenBank: KP013750–KP013757] were identified from RNAseq data. Protein alignments were constructed with MAFFT [131 (link)], and poorly aligned regions were removed with Gblocks [132 (link)]. RAxML [133 (link)] was used to infer gene orthologies (Additional file 5 : Figure S4). Resulting trees were formatted with FigTree. Single colorimetric in situ hybridization was performed as described in [34 (link)]. Fluorescent in situ hybridization of foxA in EdU-treated embryos was performed following the regular colorimetric protocol up to antibody incubation, when samples were incubated overnight with an anti-DIG POD-conjugated antibody (Roche, Indianapolis, IN, USA) diluted 1:250 in blocking solution. After extensive washes, the signal was developed with a TSA-Cy3 kit (Perkin-Elmer, Waltham, MA, USA) following manufacturer’s recommendations. The TSA reaction was stopped in detergent solution (1% Triton X-100, 1% SDS, 0.5% sodium deoxycholate, 50 mM Tris pH 8, 150 mM NaCl) at 60°C, and embryos washed several times in PTw afterwards. Subsequent fluorescent labeling of the EdU incorporation in these embryos was performed as suggested by the EdU kit manufacturer (Life Technologies).
Tsa cy3 kit
Manufactured by PerkinElmer
Sourced in United States
The TSA Cy3 kit is a laboratory product designed to facilitate the detection and visualization of target molecules in biological samples using fluorescence-based techniques. The kit contains reagents and materials necessary for the tyramide signal amplification (TSA) method, which allows for the amplification of fluorescent signals generated by Cy3 dyes. The core function of this kit is to enhance the sensitivity and detection of target analytes in various research and diagnostic applications.
Automatically generated - may contain errors
Lab products found in correlation
Glucagon, by Cell Signaling Technology (1 mentions)
Insulin, by Agilent Technologies (1 mentions)
Foxo1, by Cell Signaling Technology (1 mentions)
Dig 11 utp, by Roche (1 mentions)
Peroxidase conjugated anti dig antibody, by Roche (1 mentions)
Sp6 t7 transcription kit, by Roche (1 mentions)
Sense and anti sense rna probes, by Roche (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
T9028, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
5 protocols using tsa cy3 kit
1
Comparative Transcriptomic Sequence Analysis
2
Detailed Immunohistochemistry Protocols for Pancreatic Cells
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Our methods for sectioning, embedding, and measuring Insulin-positive area have been described previously [41] (link). Antibodies used were as follows: Nkx6.1 (Developmental Studies Hybridoma Bank #F55A12; 1:100 dilution overnight at 4 °C), Pdx-1 (Cell Signaling #5679; 1:300 dilution overnight at 4 °C), MafA (LSBio# LS-C286590; 1:75 dilution overnight at room temperature), Insulin (Dako # A0564), Foxo1 (Cell Signaling #2880; 1:50 dilution overnight at room temperature), p65 (Cell Signaling # 8242; 1:1000 dilution for 2 h at room temperature) and Glucagon (Cell Signaling #2760; 1:300 dilution overnight at 4 °C). All antibodies were detected with either Alexa Fluor secondary conjugation (Alexa 488, Alexa Fluor Plus 555), except for Nkx6.1, which was detected using a biotin/streptavidin exposure method, and, Foxo1, which was detected using a Perkin Elmer TSA Cy3 kit.
3
Fluorescent mRNA Labeling by Cytoplasmic FISH
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Fluorescent mRNA labeling by cytoplasmic fluorescence in situ hybridization (FISH) was performed as described previously [38 (link)]. The full-length coding region of mouse Fgf5 was amplified by PCR from EpiSC cDNA using following primers: mFgf5 fwd, 5’-ATGAGCCTGTCCTTGCTCTTCCTC-3’ and mFgf5 rev, 5’-TCATCCAAAGCGAAACTTCAGTCTG-3’. Digoxigenin (DIG)-11-UTP (Cat# 11209256910, Roche) -labeled antisense RNA probes were generated by T7 RNA polymerase using SP6/T7 transcription kit (Cat# 10999644001, Roche). Hybridization with DIG-labeled probes was performed overnight at 65°C. The embryos were then incubated with a peroxidase conjugated anti-DIG antibody (Cat# 11207733910, Roche, Basel, Switzerland) for 1 h at room temperature. Fluorescent staining was carried out with a Tyramide signal amplification cyanine 3 system (TSA-Cy3) kit (Code# NEL704A, Perkin-Elmer, Waltham, MA, USA) according to the manufacturer’s recommendations. To amplify the fluorescence signal, a TSA-biotin amplification kit (Code# NEL700A, Perkin-Elmer) was used.
4
Detecting dAtg1 transcripts in Drosophila
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For in situ hybridization to detect dAtg1 transcripts, Drosophila cDNA clone LD18893 (Berkeley Drosophila Genome Project expressed sequence tags, Drosophila Genomic Resource Center) was used as a template to generate digoxigenin-labeled sense and antisense RNA probes (Roche). Labeled probes were detected with a TSA Cy3 kit (PerkinElmer) as previously described [88 (link)].
5
Whole-mount in situ Hybridization and Immunohistochemistry
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Single whole-mount colorimetric and fluorescent in situ hybridization was performed following an established protocol (31) with probe concentration of 0.1 ng/μl (I. pulchra) or 1 ng/μl (the remaining species) and hybridization temperature of 67 °C. Proteinase K treatment time was adjusted for each species and ranged from 2 minutes (P. harmeri, O. fusiformis, S. californicum) to 10 minutes (T. transversa). Post-hybridization low salt washes were performed with 0.05x saline sodium citrate (SSC; H. miamia) or 0.2x SSC (the remaining species). Fluorescent in situ hybridization was visualized with TSA Cy3 kit (PerkinElmer, NEL752001KT). Samples were mounted in 70% glycerol or subjected to immunohistochemistry for visualization of neural structures: samples were permeabilized in 0,2 % Triton-x in PBS (PTx) and blocked in 1% bovine serum albumin in PTx (PBT) and incubated with antibodies against tyrosinated tubulin (Sigma, T9028) at a concentration of 1:250 in PTx with 5% normal goat serum, and incubated for 16-18 hours at 4 °C. After several washes in PBT the samples were incubated with secondary goat anti-mouse antibodies conjugated with AlexaFluor 488 (Life Technologies), at a concentration 1:200 in PTx with 5% normal goat serum for 16-18 hours at 4 °C, and samples washed extensively before mounting in 70 % glycerol and imaging. Nuclei were stained with DAPI (Molecular Probes).
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