The dissected tooth germs from the lower jaw of EGFP positive embryos were put into the Hank’s solution (Sigma Aldrich). The Hank’s solution was replaced by 1% trypsin solution (Difco Laboratories) in 4°C for one to two hours (according to the developmental stage of embryos) to dissociate the epithelium from the mesenchyme. Dissociated epithelia were documented in the Stop solution (20% FCS - Sigma Aldrich) using the inverted fluorescent microscope Leica AF6000 (Leica Microsystems GmbH, Germany). Shh expression domains were determined according to the green fluorescence in the cells actually expressing Shh.
Stop solution
Manufactured by Merck Group
Sourced in United States
The Stop solution is a laboratory reagent used to halt or stop a specific chemical reaction or enzymatic process. It is a crucial component in various analytical and experimental procedures, serving to terminate the reaction and stabilize the reaction products for further analysis.
Automatically generated - may contain errors
Lab products found in correlation
Tmb substrate, by Merck Group (3 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Af6000, by Leica (1 mentions)
Hank s solution, by Merck Group (1 mentions)
Trypsin solution, by BD (1 mentions)
Tetramethylbenzidine tmb substrate, by Merck Group (1 mentions)
Anti citrullinated histone 3, by Abcam (1 mentions)
Mastercycler realplex, by Eppendorf (1 mentions)
Beclin sirna, by Horizon Discovery (1 mentions)
Amplification grade dnase, by Merck Group (1 mentions)
Dnase, by Merck Group (1 mentions)
Nupage novex bis tris gel, by Thermo Fisher Scientific (1 mentions)
Spermine, by Merck Group (1 mentions)
Streptavidin hrp, by Merck Group (1 mentions)
Rotenone, by Merck Group (1 mentions)
Startingblock, by Thermo Fisher Scientific (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
6.5 mm transwell chamber, by Corning (1 mentions)
Maxima sybr green fluorescein qpcr master mix, by Thermo Fisher Scientific (1 mentions)
Icycler, by Bio-Rad (1 mentions)
14 protocols using stop solution
1
Isolating Shh-expressing Cells from Embryonic Tooth Germ
2
Transwell Permeability Assay for Endothelial Cells
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A Transwell permeability assay was used to assess the permeability of endothelial cells. Cells (2 × 105) in 300 uL of medium were seeded in the membrane of each 6.5-mm Transwell insert to form a fusible monolayer. Next, cells were co-cultured in a 24-well plate using the various co-culture protocols described above. After 24 h, the EC-seeded chambers were transferred to new 24-well plates, the medium above the chambers was removed, and the chambers were refilled with medium containing horseradish streptavidin peroxidase. After 5 min of incubation, 20 uL of medium was transferred from the lower chamber to a new 96-well plate and add 50 uL of TMB substrate was added to each well. After 5–20 min of incubation at room temperature, 25 uL of stop solution (Sigma) was added to well. Finally, the absorbance at 450 nm of each well was assessed with a microplate reader.
3
Quantification of Antibody Binding to cNETs
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A 96-well plate was coated with cNETs (4 μg/ml) in RPMI overnight at 4°C. Wells were washed and blocked with 1% BSA at room temperature for 1 hour. Diluted plasma (1:10) was added to the wells in (1% BSA) blocking buffer and incubated overnight at 4°C. The wells were washed three times, and donkey anti-human IgG–conjugated HRP antibody (Bio-Rad) was added to the wells in blocking buffer at 1:10,000. Wells were washed five times followed by the addition of TMB substrate (Sigma-Aldrich) and stop solution (Sigma-Aldrich). The absorbance was measured at 450 nm, and values were calculated as an OD index. Assay was performed in duplicate.
4
Quantification of Serum Citrullinated Histones and Carbamylated Protein-DNA Complexes
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Serum citrullinated histone H3 or citrullinated histone H4 or carbamylated protein–dsDNA (double-stranded DNA) complexes were quantified by ELISA. A 96-well plate was coated with rabbit polyclonal anti-citrullinated histone 3 (Abcam) or citrullinated histone 4 (EMD Millipore) or carbamylated lysine (Cell Biolabs) antibodies at 1:400 in phosphate-buffered saline (PBS) overnight at 4°C. Wells were washed and blocked with 1% bovine serum albumin (BSA) at room temperature for 1 hour. Diluted plasma (1:10) was added to the wells in (1% BSA) blocking buffer and incubated overnight at 4°C. The wells were washed three times and incubated with mouse monoclonal anti-dsDNA antibody (EMD Millipore) at 1:100 in blocking buffer. After washing three times, goat anti-mouse–conjugated horseradish peroxidase (HRP) antibody (Bio-Rad) was added to the wells in blocking buffer at 1:10,000. Wells were washed five times followed by the addition of tetramethylbenzidine (TMB) substrate (Sigma-Aldrich) and stop solution (Sigma-Aldrich). The absorbance was measured at 450 nm, and values were calculated as an optical density (OD) index. Assay was performed in duplicate.
5
Beclin-1 Silencing Impacts Total RNA Isolation
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Total RNA was isolated by Trizol reagent from DCs incubated with beclin siRNA according to the manufacturer’s instructions (Dharmacon, Chicago, IL, USA). RNA was quantified with the help of NanoDrop. A260/A280 ratio of all samples was in the range of 1.90–2.00. Intactness of RNA samples was determined with the help of formaldehyde denaturing agarose gel-electrophoresis. DNA contamination from RNA samples was removed by amplification grade DNase (Sigma, St Louis, MO, USA). Briefly, RNA samples (1 μg) were incubated with DNase (1U) for 15 min in the reaction buffer. After the incubation, DNase activity was terminated by stop solution (Sigma, St Louis, MO, USA). Furthermore, the samples were heated to 70°C for 10 min to inactivate DNase activity. Results are represented in the form of re-expression (fold) relative to untreated controls. Analysis was done by comparative Ct method, whereas Ct values were normalized against house-keeping control actin. RT-qPCR and data analysis were done by Realplex Mastercycler (Eppendorf, Hamburg, Germany).
6
In Vitro Phosphotransfer Assay
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Phosphotransfer to purified VanUG and VanRG were carried out in buffer A. The reaction was initiated by the addition of 10 μl of the purified autophosphorylation reaction mixture of VanSG (40 μg) described above to a 15 μl reaction mixture containing VanUG or VanRG (55 μg each). After incubation for various periods of times at room temperature, the phosphotransfer reactions were quenched by the addition of stop solution (Sigma) followed by electrophoresis on 12% NuPAGE Novex Bis-Tris gels (Invitrogen) in MOPS buffer (1X) and autoradiography.
7
Antioxidant Assays in Rat Models
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Diphenyl-1-picryhydrazy (DPPH), trichloroacetic acid (TCA), thiobarbituric acid (TBA), mannitol, sucrose, N-(2-hydroxyethyl) pipearizine-N-(2-ethanesulfonic acid) (HEPES), rotenone, spermine, bovine serum albumin (BSA),standard solution, standard diluent, chromagen A and B, anti-cytochrome C antibodies labelled with biotin, streptavidin-HRP, stop solution, 30X wash solution and all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO, USA) and were of the highest purity grade. Healthy male Wistar strain albino rats (weighing between 120 and 160 g), purchased from the Federal University of Agriculture, Abeokuta (FUNAAB), Nigeria were fed with standard commercial diets and water ad libitum and handled in accordance with the WHO Good Laboratory Practice (GLP) regulations throughout the experiment period.
8
Quantifying IL-10 Levels in Cell Cultures
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Using a standard ELISA protocol and matched antibody pairs, levels of IL-10 were determined in culture supernatants. Briefly, 96 well plates (Nunc, UK), were coated with 4 µg/ml of human anti-IL-10 monoclonal antibody (R&D Systems, UK). Following an overnight incubation at 4°C, plates were washed and blocked with 200 µL of StartingBlock™ (Thermo Scientific, UK) for 30 mins at room temperature. Blocking buffer was removed, and without washing, supernatants were added to the plates along with cytokine standards ranging from 4000 pg/mL to 62.5 pg/mL and incubated for a minimum of 2 hrs at room temperature. Plates were then washed and incubated with 0.5 µg/mL of anti-IL-10 biotinylated antibody (R&D Systems, UK) for 1 hr at room temperature. Plates were washed and incubated with 0.1 µg/mL of Strepavidin peroxidise (High Sensitivity, PIERCE, UK) for 30 mins at room temperature. Following a final wash, TMB substrate (Sigma, UK), was added and the reaction was stopped with Stop Solution (Sigma, UK). Plates were read using an ELISA plate reader at 450 nm.
9
Transwell Permeability Assay for Endothelial Cells
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Transwell permeability assay was used to study the permeability of ECs.25 (link) We seeded 2×105 ECs in 300 μL medium in 6.5 mm transwell chambers (Corning) and cultured the cells until the formation of a confluent monolayer. Then, the ECs were co-cultured in a 24-well plate inoculated with BMSCWT, BMSCVector, and BMSCHOXB4 and simultaneously administered with LPS. After 24 h, the chambers inoculated with ECs were transferred to a 24-well plate, the top medium of the chambers was removed and refilled with medium containing streptavidin-horseradish peroxidase. After 5 min of incubation, 20 μL of medium was collected from the lower chamber and moved to a new 96-well plate. Next, 50 µL of TMB substrate was added to each well of the 96-well plate and allowed to react for 5–20 min at room temperature, followed by the addition of 25 µL of stop solution (Sigma) to each well. Absorbance was detected at 450 nm using an enzyme linked immunosorbent assay (ELISA) reader.
10
Quantification of HSPG2 Domain Binding
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HSPG2 domains 1 and 5 were coated onto ELISA plates for 2 hours at room temperature, using a concentration of 10 µg/mL. Following three PBS washes, plates were blocked with blocking buffer (PBS, 2% v/v BSA) for 2 hours at room temperature. Tw1S4_6 IgG, diluted in blocking buffer at the indicated concentrations, was plated in triplicate and incubated overnight at 4 °C. The plates were then washed and incubated with 1:20,000 Protein-L HRP (Genscript) for 2 hours at room temperature. Three washes in PBS followed by incubation with TMB substrate (Sigma) for 15 minutes were performed. The peroxide solution was quenched with stop solution (Sigma), and signal intensity was quantified on a UV/Vis 96-well plate reader (BioTek Instruments, Vermont, USA) by measuring absorbance at 450 nm. Log concentration - response plots were generated in GraphPad Prism software. Nonlinear regression analysis was employed to generate fitted curves to the data.
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