Neutral buffered formalin (NBF), Quadrol®, triethanolamine and 4′,6-diamidino-2-phenylindole (DAPI) dilactate were purchased from Sigma Aldrich. Normal goat serum was purchased from ThermoFisher. Urea and sucrose were purchased from Chem-Supply. Triton-X-100 was purchased from VWR International. The following primary antibodies were used for immunostaining: chicken anti-GFP (Abcam, ab13970, batch #s GR3190550-3 and -12), rat anti-F4/80 (Novus, NB600-404), rat anti-keratin 8 (DSHB, TROMA-I, batch #s 7/7/16 and 30/3/17), rabbit anti-keratin 5 (BioLegend, 905504, batch # B230397) and rabbit anti-SMA (Abcam, ab5694, batch # GR3183259-26). The following secondary antibodies were used: goat anti-chicken Alexa Fluor-488 (ThermoFisher, A21236), goat anti-rat Cy3 (ThermoFisher, A10522) and goat anti-rabbit Alexa Fluor-647 (ThermoFisher, A21245).
Sucrose
Manufactured by Chem-Supply
Sourced in Australia
Sucrose is a carbohydrate compound commonly known as table sugar. It is a disaccharide composed of glucose and fructose. Sucrose is a white, crystalline solid that is widely used as a sweetener in food and beverage products.
Automatically generated - may contain errors
Lab products found in correlation
Dichloromethane, by Merck Group (2 mentions)
Fructose, by Merck Group (2 mentions)
Aniline, by Merck Group (2 mentions)
Vanillin, by Merck Group (2 mentions)
Maltose, by Merck Group (2 mentions)
Methanol, by Scharlab (2 mentions)
1 butanol, by Chem-Supply (2 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
A21236, by Thermo Fisher Scientific (1 mentions)
A 21245, by Thermo Fisher Scientific (1 mentions)
Ab13970, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole dilactate dapi, by Merck Group (1 mentions)
Normal goat serum (ngs), by Thermo Fisher Scientific (1 mentions)
Triton x 100, by Avantor (1 mentions)
A10522, by Thermo Fisher Scientific (1 mentions)
Ab5694, by Abcam (1 mentions)
Urea, by Chem-Supply (1 mentions)
5 hydroxymethylfurfural, by Thermo Fisher Scientific (1 mentions)
Anhydrous magnesium sulphate, by Scharlab (1 mentions)
4 5 7 trihydroxyflavanone, by Thermo Fisher Scientific (1 mentions)
13 protocols using sucrose
1
Immunohistochemistry Protocol for Tissue
2
Comprehensive Analysis of Commercial Honeys
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The chemicals and reagents and their suppliers were as follows: glucose, sucrose (Chem-Supply Pty Ltd., St. Gillman, SA, Australia), fructose, maltose, aniline, vanillin (Sigma-Aldrich, St. Louis, MO, USA), boric acid (Pharma Scope, Welshpool, WA, Australia), 4,5,7-trihydroxyflavanone, 5-hydroxymethylfurfural (Alfa Aesar, England, UK), DPPH* (Fluka AG, Buchs SG, Switzerland), gallic acid, diphenylamine, phosphoric acid (Ajax Finechem Pvt Ltd., Sydney, Australia), anhydrous magnesium sulphate (Scharlau, Barcelona, Spain) and Folin and Ciocalteu’s Phenol Reagent 2N (Sigma-Aldrich, St. Louis, MO, USA).
The solvents and their suppliers were as follows: methanol (Scharlau, Barcelona, Spain), 1-butanol (Chem-Supply Pty Ltd., St. Gillman, SA, Australia), 2-propanol (Asia Pacific Specialty Chemicals Ltd., Sydney, Australia), dichloromethane (Merck KGaA, Darmstadt, Germany), toluene (APS Chemicals, Sydney, Australia), ethanol, ethyl acetate and formic acid (Ajax Finechem Pvt Ltd., Sydney, Australia).
The commercial honeys (Table 6 ) were obtained from beekeepers and supermarkets in Western Australia. An artificial honey was prepared as the comparator honey (see Section 3.3.2 ).
The solvents and their suppliers were as follows: methanol (Scharlau, Barcelona, Spain), 1-butanol (Chem-Supply Pty Ltd., St. Gillman, SA, Australia), 2-propanol (Asia Pacific Specialty Chemicals Ltd., Sydney, Australia), dichloromethane (Merck KGaA, Darmstadt, Germany), toluene (APS Chemicals, Sydney, Australia), ethanol, ethyl acetate and formic acid (Ajax Finechem Pvt Ltd., Sydney, Australia).
The commercial honeys (
3
Quantification of Imidacloprid and Metabolites
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IMI (N-[1-[(6-Chloro-3-pyridyl)methyl]-4,5-dihydroimidazol-2-yl]nitramide) and [13C6]-IMI (>99% isotopic purity; >97% total purity) were obtained from AK Scientific (Union City, CA, USA) and IsoSciences (King of Prussia, PA, USA), respectively. Authentic standards of IMI-Ole (N-[1-[(6-chloro-3-pyridyl)methyl]-1,3-dihydro-2H-imi-dazol-2-ylidene]nitramide) and IMI-5-OH (N-(1-[(6-chloro-3-pyridyl)methyl]-5-hydroxyimidazolidin-2-ylidene]nitramide) were provided by Bayer CropScience AG (Monheim, Germany). Sucrose was obtained from Chem-Supply (Gillman, SA, Australia) and glacial formic acid from AnalaR (supplied by BDH, Poole, Great Britain). HPLC grade acetonitrile, ethyl acetate,and HPLC grade methanol were obtained from Merck (Kenilworth, NJ, U.S.A.). 18.2 M ℧ HPLC grade water was obtained from Honeywell (Morristown, NJ, USA).
4
RNA Solution Preparation and Characterization
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For RNA solution (10 mg/mL, ribonucleic acid
from torula yeast, Type VI, Sigma-Aldrich), 100 mg of yeast RNA was
added to 10 mL of 10 mM EDTA solution (ChemSupply Australia) in Milli-Q
water. The RNA solution was adjusted to pH 6 with 5 M NaOH (Lowy Solutions).
The QuantiFluor RNA System (Promega) was used as the RNA dye; other
dyes used include 1 mM pyranine (Sigma-Aldrich) as an encapsulation
marker, 0.1 mM acridine orange as a commonly-used cationic dye, and
5 μm Rhodamine B (Sigma-Aldrich) as a membrane dye. Sucrose
(ChemSupply Australia) at 0.1 M was also used as a neutral encapsulation
molecule. Buffers used include 1× PBS made from 10× PBS
stock solution (Lowy Solutions) and 0.01 M citrate buffer (ChemSupply
Australia), with pH adjusted with 5 M NaOH or HCl (Lowy Solutions).
NaCl solutions (10 mM) were made by appropriate dilutions of 5 M NaCl
solution (Lowy Solutions). pH was measured using an Orion Star A121
pH meter with an Orion 8103BN ROSS probe.
from torula yeast, Type VI, Sigma-Aldrich), 100 mg of yeast RNA was
added to 10 mL of 10 mM EDTA solution (ChemSupply Australia) in Milli-Q
water. The RNA solution was adjusted to pH 6 with 5 M NaOH (Lowy Solutions).
The QuantiFluor RNA System (Promega) was used as the RNA dye; other
dyes used include 1 mM pyranine (Sigma-Aldrich) as an encapsulation
marker, 0.1 mM acridine orange as a commonly-used cationic dye, and
5 μm Rhodamine B (Sigma-Aldrich) as a membrane dye. Sucrose
(ChemSupply Australia) at 0.1 M was also used as a neutral encapsulation
molecule. Buffers used include 1× PBS made from 10× PBS
stock solution (Lowy Solutions) and 0.01 M citrate buffer (ChemSupply
Australia), with pH adjusted with 5 M NaOH or HCl (Lowy Solutions).
NaCl solutions (10 mM) were made by appropriate dilutions of 5 M NaCl
solution (Lowy Solutions). pH was measured using an Orion Star A121
pH meter with an Orion 8103BN ROSS probe.
5
Phytochemical Characterization of Natural Compounds
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Chemicals and reagents, and their sources: Glucose, Sucrose, Sodium carbonate anhydrous (Chem-Supply Pty Ltd., St. Gillman, SA, Australia), Fructose, Maltose, Aniline, Vanillin (Sigma-Aldrich, St. Louis, MO, USA), Boric acid (Pharma Scope, Welshpool, WA, Australia), 4,5,7-trihydroxyflavanone and 5-hydroxymethylfurfural (Alfa Aesar, England, UK), DPPH* (Fluka AG, Buchs SG, Switzerland), Gallic acid, Diphenylamine, Phosphoric acid, 3,4,5-trihydroxybenzoic acid (Ajax Finechem Pvt Ltd., Sydney, NSW, Australia), Anhydrous sodium sulfate (Merck KGaA, Darmstadt, Germany), Folin and Ciocalteu’s Phenol Reagent 2N (Sigma-Aldrich, St. Louis, MO, USA).
Solvents and their sources: Methanol (Scharlau, Barcelona, Spain), 1-Butanol (Chem-Supply Pty Ltd., St. Gillman, SA, Australia), 2-Propanol (Asia Pacific Specialty Chemicals Ltd., Sydney, Australia), Dichloromethane (Merck KGaA, Darmstadt, Germany), Toluene (APS Chemicals, Sydney, NSW, Australia), Ethanol, Ethyl acetate and Formic acid (Ajax Finechem Pvt Ltd., Sydney, NSW, Australia).
Solvents and their sources: Methanol (Scharlau, Barcelona, Spain), 1-Butanol (Chem-Supply Pty Ltd., St. Gillman, SA, Australia), 2-Propanol (Asia Pacific Specialty Chemicals Ltd., Sydney, Australia), Dichloromethane (Merck KGaA, Darmstadt, Germany), Toluene (APS Chemicals, Sydney, NSW, Australia), Ethanol, Ethyl acetate and Formic acid (Ajax Finechem Pvt Ltd., Sydney, NSW, Australia).
6
Cryosectioning Wound Tissue Samples
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Mice were culled using CO2 asphyxiation. Organs and 6 mm wounds with 2 mm of surrounding skin were dissected and fixed in 4% paraformaldehyde (Sigma-Alrich, MO, USA) for 2 h at 4 °C. Fixed samples were cyro-protected with incubations in 10% and subsequently 30% sucrose (Chem-Supply, SA, AUS) in phosphate buffered solution (PBS) overnight. The samples were embedded in Tissue-Tek Optimal Cutting Temperature (Sakura Finetek, CA, USA) in preparation for cyro-sectioning. Before embedding, wounds were cut in half to ensure that sections were taken from the centre of the granulation tissue, whereby 10 µM sections were taken at 30 µM intervals onto Superfrost Plus slides (Thermo Fisher Scientific, Waltham, USA) using a CM1905 Cyrostat (Leica Biosystems, Wetler, Germany). Slides were stored at −30 °C.
7
Brain Fixation and Sectioning Protocol
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At 7d or 35d post-injury mice were anaesthetised with 300μl of equal parts Lethabarb (Sodium Pentobarbitone; Virbac, Sydney, Australia) and saline and perfused through the left ventricle with 0.1M Phosphate Buffered Saline (20mL) to flush out the blood, followed by 4% paraformaldehyde (PFA; Sigma) in PBS (20mL) for fixing the tissue. The brain was dissected out and placed in 4% PFA at 4°C overnight followed by 30% sucrose (Chem-Supply Pty Ltd.) in PBS at 4°C for 48h.
Brains were hemisectioned with the aid of a mouse brain blocker (David Kopf Instruments, Sydney, NSW). The coronal cut was made at slot number 5 such that the front half of the tissue block contained lateral ventricles and the other half the hippocampus. Brains were placed cut side down into plastic Tissue-Tek® Cryomolds (Grale Scientific Pty Ltd., Australia), covered in in Tissue-Tek® optimum cutting temperature compound (O.C.T.; Grale Scientific Pty Ltd.), frozen in isopentane cooled over dry ice and stored at −80°C until use.
Brains were hemisectioned with the aid of a mouse brain blocker (David Kopf Instruments, Sydney, NSW). The coronal cut was made at slot number 5 such that the front half of the tissue block contained lateral ventricles and the other half the hippocampus. Brains were placed cut side down into plastic Tissue-Tek® Cryomolds (Grale Scientific Pty Ltd., Australia), covered in in Tissue-Tek® optimum cutting temperature compound (O.C.T.; Grale Scientific Pty Ltd.), frozen in isopentane cooled over dry ice and stored at −80°C until use.
8
Sectioning and Preservation of Brain Tissue
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Sectioning of brain tissue was done using a Leica cryostat. All free floating sections were collected in 24 well plates containing 500μl PBS. Free floating serial coronal sections, 30μm thick were collected through the injury site (bregma −1.2mm to 1.20mm). For each animal, 9 wells containing 6–8 sections spaced 300μm apart were collected. Every 10th consecutive section was collected onto a Superfrost® Plus slide (Grale Scientific Pty Ltd.) for Hemotoxylin and Eosin staining. Following collection of all free floating sections, PBS was replaced with 500μl of anti-freeze (15% sucrose (Chem-Supply Pty Ltd.) and 30% Ethylene Glycol (Chem-Supply Pty Ltd.) in PBS) and sections stored at −20°C until staining.
9
Optic Nerve Transection in Rats
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Three days after partial optic nerve transection, rats were euthanised with pentobarbitone sodium (160 mg/kg, Delvet). Rats in cohort 1 were transcardially perfused with 0.9% saline, followed by 4% paraformaldehyde (PFA; Sigma-Aldrich) in 0.1 M phosphate buffer. Optic nerves were dissected and fixed overnight in 4% PFA and subsequently transferred into 15% sucrose (Chem Supply), 0.1% sodium azide (Sigma-Aldrich) in PBS pH 7.2 for storage. Tissue was then cryosectioned longitudinally to a thickness of 14 µm and collected onto Superfrost Plus glass microscope slides. In cohort 2, blood samples were taken via cardiac puncture from the rats following euthanasia. Blood samples were collected into heparinised tubes and spun at 3000 rpm, 4 °C for 10 minutes. Plasma was separated and stored at −80 °C until subsequent multiplex assay analysis.
10
Spinosad Insecticide Exposure Survival
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For all tests 5 replicates of 20 individuals (100 individuals) per condition were used. In assessing third instar larval viability and metamorphosis following insecticide exposure, individuals were rinsed three times with 5% w/v sucrose (Chem Supply) and placed in vials on insecticide-free food medium.
Survival probability of larvae exposed to 2.5 ppm spinosad for 2 hr was analysed using Kaplan-Meier method and the Log-rank Mantel-Cox test. Correct percentage survival of larvae exposed to 0.5 ppm spinosad for 2 hr, or 0.1 ppm spinosad for 4 hr was analysed using Abbots' correction. To examine the survival of adult flies chronically exposed to 0.2 ppm spinosad, 5 replicates of 20 females (3-5 days old) were exposed for 25 days. The same number of flies was used for the control group.
Statistical analysis was based on the Kaplan-Meier method and data were compared by the Log-rank Mantel-Cox test.
Survival probability of larvae exposed to 2.5 ppm spinosad for 2 hr was analysed using Kaplan-Meier method and the Log-rank Mantel-Cox test. Correct percentage survival of larvae exposed to 0.5 ppm spinosad for 2 hr, or 0.1 ppm spinosad for 4 hr was analysed using Abbots' correction. To examine the survival of adult flies chronically exposed to 0.2 ppm spinosad, 5 replicates of 20 females (3-5 days old) were exposed for 25 days. The same number of flies was used for the control group.
Statistical analysis was based on the Kaplan-Meier method and data were compared by the Log-rank Mantel-Cox test.
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