IMR90 fibroblasts were plated and treated with ActD or DMSO as previously described (see section Inhibition of transcription). After fixation with 4% PFA, cells were prepared for OligoSTORM as previously described (Beliveau et al., 2017 (link)). Briefly, cells were permeabilized with 0.5% Triton X-100 in PBS for 10 min and with 0.1% Tween in PBS for 2 min at room temperature. Cells were then incubated for 5 min in 0.1 N HCl and washed twice with 2x SSCT for 1 min each, followed by a 5 min wash at room temperature in 2xSSCT - 50% Formamide (Sigma-Aldrich, #F9037) and stored for up to one week in 2x SSCT - 50% Formamide at 4°C or used immediately for imaging. Before hybridization, the samples were incubated in 2x SSCT - 50% Formamide at 60°C for 20 min. Then, the hybridization mix is prepared (2x SSCT, 50% Formamide, 10% (w/v) dextran sulfate (Sigma-Aldrich, #S4030), 0.4 μg/μL of RNase A (Thermo Fisher Scientific, #EN0531), 50 pmoles of 5′ AF405 labeled primary probes and 1 μL 100 μM 3′ AF647 labeled secondary probes) (see key resources table and Table S5 ), added to the samples, denatured at 78°C for 3 min and subsequently incubated at 42°C for 16 h. Samples were washed twice with 2x SSCT at 60°C for 10 min each, once with 2x SSCT for 10 min at room temperature and once with 2x SSC.
Formamide
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China, Poland, Israel, France, Ireland, Canada, Spain, Japan
Formamide is a colorless, odorless, and hygroscopic liquid. It is a common laboratory solvent used in various chemical and biological applications. Formamide has a high boiling point and is miscible with water and many organic solvents.
Automatically generated - may contain errors
Lab products found in correlation
Evans blue dye, by Merck Group (52 mentions)
Evans blue, by Merck Group (49 mentions)
Dextran sulfate, by Merck Group (17 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (10 mentions)
Ethylene glycol, by Merck Group (9 mentions)
Formamide, by Thermo Fisher Scientific (9 mentions)
Diiodomethane, by Merck Group (8 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (7 mentions)
Triton x 100, by Merck Group (6 mentions)
Glycerol, by Merck Group (6 mentions)
Urea, by Merck Group (6 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (6 mentions)
Trizol reagent, by Thermo Fisher Scientific (6 mentions)
Eb dye, by Merck Group (5 mentions)
Paraformaldehyde (pfa), by Merck Group (5 mentions)
Sodium chloride (nacl), by Merck Group (5 mentions)
E2129, by Merck Group (4 mentions)
Yeast trna, by Thermo Fisher Scientific (4 mentions)
Blocking reagent, by Roche (4 mentions)
388 protocols using formamide
1
Single-Molecule RNA Imaging in Fibroblasts
2
Tissue Clearing Protocol for Microscopy
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Samples were successively immersed in: (i) a 30% triethanolamine (TEA, Sigma)/40% formamide (Sigma) solution (15 min for slices and for EBs, 20 min for brain organoids, 3 h 20 min for a whole E10.5 embryo); (ii) a 60% TEA/25% formamide solution (25 min for slices and for EBs, 30 min for brain organoids, 5 h for embryo); and (iii) a 70% TEA/15% formamide solution (25 min for slices and for EBs, 30 min for brain organoids, 5 h for whole embryo). In either case, after clearing, samples were mounted under a glass coverslip in a chamber filled with the respective final solution.
3
Quantifying Vascular Permeability via Evans Blue
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A 0.5% sterile solution of Evans blue (Sigma, Ireland) was prepared in PBS and 200 μl slowly injected into the tail vein of mice. After 30 min the mice were sacrificed through cervical dislocation and organs collected and weighed. 500 μl formamide (Sigma, Ireland) was added to the organs and heated to 55°C in a heat block for 24 h. The formamide/Evan's blue mixture was centrifuged to pellet any tissue and the supernatant measured at 610 nm, using formamide as a blank. The amount of Evan's blue extravasated per mg of tissue was then calculated as outlined in (39) (link).
4
Evaluation of Blood-Brain Barrier Permeability
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To measure BBB permeability, Evans blue (EB) dye was used as a tracer, as previously described (27 (link)). Briefly, the rats were injected with 2% EB solution diluted in normal saline through the tail vein (2 ml/kg of body weight; Sigma-Aldrich; Merck KGaA) 24 h post-surgery. The EB dye was allowed to circulate for 2 h. Next, the rats were anesthetized and transcardially perfused using 0.9% sodium chloride. The brains were removed, divided into left and right hemispheres, then the right hemisphere was immersed in formamide (10 ml/kg; Sigma-Aldrich; Merck KGaA) at 60˚C for 24 h. Cortical proteins were formamide extracted and centrifuged (2,500 x g) for 10 min at 4˚C. A total of 1 ml supernatant was measured in a spectrophotometer at 620 nm to compare EB content in the brain tissue with standard EB solution.
5
Senescence-Associated β-Galactosidase Staining Protocol
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Following the SA-β-gal staining protocol, cells were fixed with 2% PFA solution for 10 min at room temperature, washed, and dehydrated in prechilled 70% ethanol for 3 h at 4 °C. Thereafter, cells were washed in PBS RNAse free for 10 min, incubated in 40% formamide (Millipore, Ca) in 2× saline-sodium citrate (SCC) buffer (300 mM sodium chloride, 30 mM sodium citrate, pH 7.0) for 10 min at room temperature, and blocked in hybridization buffer (40% formamide, 2× SCC buffer, 200 μg/mL bovine serum albumin, 100 mg/mL dextran sulfate, 2 mM vanadyl sulfate, 1 mg/mL yeast tRNA) for 15 min at 37 °C99 (link). The Cy5-labeled (CAG)10 DNA probe was denatured for 10 min at 100 °C, chilled on ice for 10 min, then added to prechilled hybridization buffer for a final concentration of 150 ng/μL probe. Cells were incubated with the probe for 2 h at 37 °C. As a negative control, RNase solution were incubated for 10 min at RT before the hybridization of the probe. Cells were washed, counterstained with DAPI, and images were acquired on SP8 confocal microscope (Leica).
6
RNA Localization Assay using MS2-Cy3 Probe
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Approximately 24 h after transfection, cells were fixed with 4% paraformaldehyde in PBS for 30 min at room temperature, washed twice with PBS, and permeabilized in 70% ethanol overnight at 4°C. The cells were rehydrated in PBS and in SSC buffer containing 20% formamide (Sigma) and incubated overnight at 37°C with hybridization mix (1× SSC, 20% formamide, 10% dextran sulfate, 0.01 µg/µL RNA grade BSA, 2 mM Vanadyl-R-C, 20 µg of tRNA, and 20 ng of MS2-Cy3 probe). The probe was briefly denaturated at 90°C before being added to the hybridization mix. Post-hybridization washes were performed twice in SSC containing 20% formamide for 30 min each at 37°C. Finally, the cells were washed in PBS for 2 min and then mounted using a mix containing 90% glycerol, 1 mg/mL p-phenylenediamine and 0.1 µg/mL DAPI. The following MS2 probe was used (X stands for amino-allyl-T): MS2 transcribed strand 5′-AXGTCGACCTGCAGACAXGGGTGATCCTCAXGTTTTCTAGGCAATXA-3′.
7
In Vivo Assessment of BBB Permeability
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To evaluate BBB permeability, mice were injected intravenously with 2% EB (Sigma-Aldrich) at a dose of 4 ml/kg. One hour after the injection, the mice were anaesthetized and perfused with normal saline (20 ml) through the left ventricle. Hippocampal tissue was extracted and immersed in formamide (Sigma-Aldrich) at 37°C for 72 h. Then, formamide was centrifuged at 12000 g for 20 min. The absorbance of was measured at 632 nm (BioTek, Winooski, Vermont, USA), and the EB content was calculated from the standard EB curve to measure BBB permeability.
8
Quantifying Brain Barrier Disruption
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BBB disruption was assessed by measuring the amount of Evans blue extravasation. Briefly, mice were intravenously injected with 0.1 ml 4% Evans blue (Sigma‐Aldrich). After 60 min, the animals were perfused transcardially with phosphate‐buffered saline (PBS) to remove intravascular Evans Blue. The brains were homogenized in formamide (Sigma‐Aldrich) and incubated in the dark at 60°C for 24 h. The absorbance of eluted Evans blue dye in formamide solution was measured using a spectrophotometer at 610 nm and normalized to plasma levels.
9
Detecting RNA Foci in Astrocytes
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Astrocytes on glass coverslips were fixed with 4% paraformaldehyde (Agar Scientific) for 15 min at room temperature followed by permeabilization in 70% ethanol at 4°C overnight. Cells were then re‐hydrated in 50% formamide (Sigma)/2x SSC (Sigma) for 10 min at room temperature and blocked in hybridization buffer (50% formamide (Sigma), 2×SSC (Sigma), 10% Dextran Sulfate (Millipore), 1 mg/ml Yeast tRNA (Invitrogen) and 1 mg/ml Salmon Sperm DNA (Invitrogen)) for 30 min at 45°C. 50 ng of an Alexa Fluor® 546‐conjugated (GGCCCC)4 probe (IDT) diluted in the hybridization buffer was applied on cells for 2 hours at 45°C in a humidified chamber. After the hybridization, cells were washed twice with 50% formamide/2× SSC for 30 min at 45°C and then once with 2× SSC for 30 min at room temperature. After another three washes with PBS at room temperature, immunofluorescence imaging was performed as described previously.
As controls, cells were treated with either 3 U/ml DNase (Life Technologies) or 100 μg/ml RNase (Sigma) diluted in 2x SSC for 1 hour at 37°C prior to the hybridization step. In addition, an anti‐sense RNA probe against the CCTG repeat expansion was also applied on cells to assess the specificity of the (GGCCCC)4 probe.
As controls, cells were treated with either 3 U/ml DNase (Life Technologies) or 100 μg/ml RNase (Sigma) diluted in 2x SSC for 1 hour at 37°C prior to the hybridization step. In addition, an anti‐sense RNA probe against the CCTG repeat expansion was also applied on cells to assess the specificity of the (GGCCCC)4 probe.
10
Quantifying Interstitial Fluid Drainage in Mice
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Drainage of interstitial fluids was quantified using Evans Blue injections in the footpad of WT and Cx43+/− mice, as described previously (Meens et al., 2017 (link)). In brief, mice were anesthetized and injected with 5 μl 5% Evans Blue (dissolved in PBS) in the left footpad using a micro syringe. After 15 min, blood was collected by puncturing the left ventricle and centrifuged 15 min at 5,000 rpm (4°C). Formamide (Sigma-Aldrich, St. Louis, MO) was added to each serum sample (500 μL Formamide/200 μL serum) and the mix was incubated overnight at 55°C. Thereafter, presence of Evans Blue in the serum was quantified by measuring the fluorescence using a SpectraMax Paradigm Multi-Mode Microplate reader (excitation: 620 nm; emission 670 nm; Molecular Devices, Sunnyvale, CA).
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