Prior to applying FL-Htt-MO, 5′-TATTGCCTTTGAGATAAATCTTCAT-carboxyfluorescein-3′ was diluted in Endo-Porter PEG (Gene Tools, LLC. Philomath, OR 97370, USA) after removing the fertilization envelope to ensure access to the cells, as follows. Fresh eggs were obtained by injecting 0.5 M KCl into the coelomic cavity, then were pretreated with 1 mmol/L 3-Amino-1,2,4-triazole (Tokyo Chemical Industry Co., Ltd. Tokyo, Japan) for 10 min, and inseminated [51 (link)]. Then, the softened fertilization envelopes were removed by passing the eggs through a 62 µm nylon mesh (Kyoshin Ricoh Inc., Tokyo, Japan). The denuded fertilized eggs were incubated in a mixture of 10 or 20 µM FL-Htt-MO [2 µL Stock sol + 4 µL Endo-Porter-PEG in 1 mL FSW (Gene Tools, LLC, https://www.gene-tools.com/content/getting-morpholinos-cultured-cells ) (Access on: 3 March 2021)] or in normal FSW in a 24-well plate at a concentration of about 10 eggs/2 mL in a well at 18 °C until 4aPL. At the sBL, 2aPL, and 4aPL stages, they were used for examining ciliary beating activity as will be described below. Aliquots of these 4aPLs were fixed for whole-mount immunohistochemical analysis, as described above.
Endo porter peg
Manufactured by Gene Tools
Endo-Porter PEG is a lab equipment product designed for the delivery of biomolecules into cells. It facilitates the intracellular delivery of a variety of macromolecules, including proteins, peptides, and oligonucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Polyvinylidene fluoride membrane, by Bio-Rad (2 mentions)
Pre cast 4 15 polyacrylamide gels, by Bio-Rad (2 mentions)
Goat anti mouse hrp, by Agilent Technologies (1 mentions)
Goat anti rabbit hrp, by Cell Signaling Technology (1 mentions)
Sc 7298, by Santa Cruz Biotechnology (1 mentions)
Ecl substrate kit, by GE Healthcare (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Ab3280, by Abcam (1 mentions)
6 protocols using endo porter peg
1
Microinjection of Fluorescent Huntingtin Morpholino
2
Oligonucleotide Synthesis and Delivery
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PMOs were synthesized by Gene Tools, LLC, and delivered into cells using Endo‐Porter (PEG) (Gene Tools, LLC). Sequences used are listed in Table EV2 .
3
Fst Knockdown in Chick Somite Tissue
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Fst-expressing somite tissue from wild-type chick embryos was dissected and cultured in Dulbecco's modified Eagle medium, 10% foetal bovine serum and 1% penicillin/streptomycin for 4 h before transfecting with 1 mM translation-blocking FstMO (Gene Tools; sequence 5′-GATCCTCTGATTTAACATCCTCAGC-3′) or 1 mM StdMO negative control (Gene Tools; sequence 5′-CCTCTTACCTCAGTTACAATTTATA-3′) using Endoporter PEG (Gene Tools). Protein was extracted after 48 h. Protein lysate (30 μg) was run on pre-cast 4-15% polyacrylamide gels (Bio-Rad) and blotted onto polyvinylidene fluoride membrane (Bio-Rad). Primary antibody against Fst (Abcam ab47941; 1:2000) was applied at 4°C overnight and secondary polyclonal goat anti-rabbit-HRP (Cell Signaling Technology, 7074; 1:2000) was applied for 1 h at room temperature. Primary antibody against HSC70 (Santa Cruz, sc-7298; 1:2500) was applied at 4°C overnight and secondary polyclonal goat anti-mouse-HRP (Agilent, P0447; 1:1000) was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit and imaged.
4
Modulating PKM and HDAC1 in Enteric Neuron Development
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Translation-blocking morpholino against HDAC1 and splice-modifying morpholinos targeting PKM: PKM1-MO (targeting splice junction between intron 8 and exon 9 of PKM) and PKM2-MO (targeting splice junction between exon 10 and intron 10 of PKM), were transfected into FACS-ENCCs with Endo-Porter PEG (Gene Tools) according to the manufacturer’s instructions. In brief, ENCCs was replenished with fresh culture medium (N2 medium containing 10 ng ml−1 FGF2 and 3 μM CHIR99021) containing 5 μM morpholinos (HDAC1-MO, PKM1-MO, PKM2-MO, or control-MO) and swirled gently. Then, 6 μl Endo-Porter PEG was added to every 1 ml of medium and swirled immediately to mix. Neuronal differentiation was started 1 day after transfection, the medium was replaced with N2 medium supplemented with GDNF (10 ng ml−1) and ascorbic acid (200 μM) and maintained for 5 days. For seahorse analysis, the cellular metabolisms of ENCCs were assayed 48 h after transfection. The oligonucleotide sequences of morpholinos are listed in Supplementary Table 3 .
5
RT-PCR and Doxo Resistance Assay
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For RT-PCR and RT-qPCR analysis, MCF7-DoxoR cells were seeded at 300 000 cells/well in a six-well plate. Twenty four hours after seeding, splice-switching oligonucleotide (SSO) or standard control morpholino (Gene Tools, sequences in Supplementary Table S1 ) were transfected at a final concentration of 5 μM with Endo-Porter PEG (Gene Tools) according to the manufacturer's instruction. Cells were harvested 48 h later for RNA extraction. For WST-1 assay, MCF7-DoxoR cells were seeded at 6000 cells/well in six replicates in a 96-well plate on day 1. Twenty four hours later on day 2, cells were transfected with 5 μM SSO or control morpholino. On day 3, triplicates were treated or not with 150 μM Doxo. Seventy two hours later on day 6, WST-1 assay was performed.
6
Quantification of Gli3 Protein in Somites
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Somites of wild-type embryos were dissected and cultured in Dulbecco's modified Eagle medium, 10% fetal bovine serum, 1% penicillin/streptomycin for 4 h before being transfected with Gli3 MO (1 mM) or control MO (1 mM) using Endoporter PEG (Gene Tools) and protein extracted after 48 h. Somites from AM133- or AMscr-injected embryos were dissected for protein extraction. Protein lysate (31.5 μg) was run on pre-cast 4-15% polyacrylamide gels (Bio-Rad) and blotted onto polyvinylidene fluoride membrane (Bio-Rad). Primary antibody against Gli3 (1:200, 6F5 Gli3N, Genentech, Wen et al., 2010 (link)) was applied at 4°C overnight before secondary polyclonal goat anti-mouse HRP (1:1000, P0447, DAKO) was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit (GE Healthcare) and imaged. Primary antibody against actin (1:1000, ab3280, Abcam) was applied at 4°C overnight; secondary polyclonal goat anti-mouse HRP was applied for 1 h at room temperature. The blots were treated with an ECL substrate kit and imaged. Quantification of blots was performed using ImageJ.
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