The preparation of biological specimens for scanning electron microscopy (SEM) observation was carried out at room temperature according to a procedure described previously by Fischer et al. (54 (link)). Treatment and control coupons were fixed with 2.5% glutaraldehyde (catalog number G6257; Sigma-Aldrich) in 0.2 M sodium cacodylate (pH 7.4) (catalog number C0250; Sigma-Aldrich) for 30 min, rinsed with 0.2 M sodium cacodylate, postfixed with 2% osmium tetroxide (OsO4) (catalog number 75632; Sigma-Aldrich) in 0.2 M sodium cacodylate for 30 min, and rinsed again with 0.2 M sodium cacodylate. Subsequently, the fixed samples were dehydrated with a graded ethanol series (30 to 100% at 10% intervals for 10 min each) and postdried with hexamethyldisilazane (HMDS) (catalog number 440191; Sigma-Aldrich) for 20 min. The dried samples were mounted onto SEM specimen stubs using conductive double-sided carbon tapes (catalog number 77825-12; Electron Microscopy Sciences, Hatfield, PA) and coated with 10 nm of gold (108auto sputter coater; Cressington Scientific Instruments, UK). Electron micrographs were obtained at an acceleration voltage of 2 kV using a Magellan 400 XHR-SEM instrument (FEI Company, Hillsboro, OR) at the Electron Microscopy Facility, University of Massachusetts—Amherst.
Sodium cacodylate
Manufactured by Merck Group
Sourced in United States, Germany
Sodium cacodylate is a chemical compound commonly used as a buffering agent in various laboratory applications. It serves to maintain a specific pH level in solutions, ensuring optimal conditions for various biological and chemical processes. The core function of sodium cacodylate is to provide a stable and controlled environment for experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Glutaraldehyde, by Merck Group (37 mentions)
Paraformaldehyde (pfa), by Merck Group (11 mentions)
Osmium tetroxide, by Merck Group (6 mentions)
Potassium ferrocyanide, by Merck Group (5 mentions)
Bovine serum albumin (bsa), by Merck Group (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Methanol, by Merck Group (4 mentions)
Auriga crossbeam system, by Zeiss (4 mentions)
Hexamethyldisilazane, by Merck Group (3 mentions)
Phosphate buffered saline (pbs), by Merck Group (3 mentions)
Hydrochloric acid (hcl), by Merck Group (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Tannic acid, by Merck Group (3 mentions)
Osmium tetroxide, by Electron Microscopy Sciences (3 mentions)
Gluteraldehyde, by Electron Microscopy Sciences (3 mentions)
Uranyl acetate, by Merck Group (3 mentions)
Peg 8000, by Merck Group (3 mentions)
Hepes, by Merck Group (3 mentions)
Polynucleotide kinase, by New England Biolabs (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
86 protocols using sodium cacodylate
1
Specimen Preparation for SEM Imaging
2
Evaluating Cellular Morphology on Anodized Materials
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To evaluate morphological changes in MC3T3-E1 cells seeded on the anodized materials, FE-SEM analysis was carried out as described by others.25 (link) After culturing for periods of 4 h, 24 h, and 21 days of seeding, cells grown on the samples were rinsed three times with PBS (5 min) and fixed in 2.5% w/v glutaraldehyde (Sigma-Aldrich, USA) buffered with 0.1 M sodium cacodylate (Sigma-Aldrich, USA) at 4 °C overnight, washed three times for 5 min in 0.1 M sodium cacodylate buffer and postfixed with 2.5% glutaraldehyde for 2 h at room temperature (RT). The cells were dehydrated in a graded series of ethanol solutions (25%, 50%, 70%, and 100%) for 15 min at each concentration. Finally, the samples were sputter-coated with gold (10-nm gold layer) for 8 s and observed at 5 kV accelerating voltage. The image J software was used to compute the length and density of filopodia on the NTs under the different biochemical serum conditions.37 (link)
3
Quantifying Microbial Biofilm Adhesion on Titanium
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After 30 days, the biofilm adhesion on the Ti sheets surface were identified under scanning electron microscopy using a MAICE system (JEOL JSM-6400 Scanning Electron Microscope, JEOL LTD, Tokyo, Japan). The monomicrobial biofilm adhering to the samples was fixed in a solution of 3% glutaraldehyde (50 wt.% in H2O, CAS#111-30-8, Sigma-Aldrich), 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich), and 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich) for 45 min. Samples were placed in a buffer solution of 0.1 mol/L sucrose (CAS#57-50-1, Sigma-Aldrich) and 0.1 mol/L sodium cacodylate (CAS#6131-99-3, Sigma-Aldrich) for 10 min. Ti sheets surface and biofilm were treated in serial ethanol dehydrations for 10 min each and dehydrated in hexamethyldisilazane (HDMS, CAS# 999-97-3, Sigma-Aldrich) until SEM imaging. Ti sheets were then sputter-coated with a palladium–gold alloy (Polaron SC 7620 Sputter Coater, Quorum Technologies, Laughton, East Sussex, UK) with a thickness of 10 nm to reduce charging effects during SEM analysis (10–15 mA, under a vacuum of 130 mTorr). After this, the SEM was operated at 5 kV, spot 3 to 6.
In addition, SEM images of the biofilm on the surface of the Ti sheets were analyzed by ImageJ software. Bacteria coverage percentages were averaged over five random areas.
In addition, SEM images of the biofilm on the surface of the Ti sheets were analyzed by ImageJ software. Bacteria coverage percentages were averaged over five random areas.
4
RNA Quantification and Characterization
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All synthetic duplex RNA samples were ordered as 10 or 20-mers in dialyzed lyophilized powdered form from Bioneer Korea Inc. and re-suspended in a suitable amount of TDW at pH 7 as per the user manuals followed by storage at − 20 °C. 100-mer poly AU RNA was purchased from Santa Cruz Biotechnology and handled in the same way. HeLa and HCT116 RNAs were extracted with TRIsure (Bioline) following the manufacturer’s instruction. The mass of extracted RNA was measured using Nanodrop and the concentration was calculated using specific extinction coefficients at 260 nm (poly AU: ~ 18,000; poly GC: ~ 13,000) for synthetic samples. For cellular RNA samples, an approximate extinction coefficient for double-stranded RNA (~ 15,000) was used for all samples. A previous study has shown that the shifts and error in extinction coefficients for ssRNA and dsRNA mixtures to be in the range of about 3–4% [66 (link)]. Based on the percent changes observed when MC was incubated with the enriched dsRNAs, the fraction and expression of dsRNAs in the sample was calculated. Sodium cacodylate buffer was prepared by dissolving Sodium cacodylate (Sigma Aldrich) in TDW and adjusting pH to 7.0 by adding diluted HCl. All chemicals were ordered from Sigma Aldrich or Tokyo Chemical Company.
5
Ultrastructural Analysis of Extracellular Matrix
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After 14 days of culture, the cell loaded samples were fixed with 2.5% glutaraldehyde (Sigma Aldrich) in 0.1 M Sodium Cacodylate (Sigma Aldrich) buffer for 15 min and washed with 0.1 M Sodium Cacodylate buffer. Control samples without cells and cell loaded samples were dehydrated using 0.5 ml of multiple ethanol series (50% twice, 70% twice, 95% twice, 100% three times for 10 min) and were dried chemically with hexamethyldisilazane (Sigma Aldrich). Then the samples were washed three times with 0.5 ml ultrapure water for 5 min, dried by air and mounted on specimen stubs. To provide better contrast, only the samples for scanning electron microscopy (SEM) imaging were sputter coated with 8 nm gold (Q3150T, Quorum Technologies). SEM images were obtained using a Quanta 600 SEM (Thermo Scientific Breda, The Netherlands), in a high vacuum (<1.3 × 10−4) at 10 kV with a spot size of 3 using the Everhart‐Thornley secondary electron detector (ETD‐SE). Similar sample preparation was utilized to perform energy dispersive x‐ray (EDX) (Phenom ProX Desktop, ThermoFisher) analysis to evaluate regions in which extracellular matrix depositions were identified (10 kV, backscattered electron detector).
6
Scanning Electron Microscopy of Juvenile Parasites
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Juveniles grown in 50% CS in RPMI were fixed in 4% glutaraldehyde (Sigma-Aldrich), for 4 h at 4°C. Following this, juveniles were washed in 0.1 M sodium cacodylate buffer (0.1 M sodium cacodylate (Sigma-Aldrich) buffer (pH 7.4), containing 3% sucrose (Sigma-Aldrich)) overnight (~16 h) at 4°C. Juveniles were then stained in 1% OsO4 (90 min, 4°C), followed by three 15 min washes in H2O at RT. Juveniles were then washed twice in 70% ethanol for 30 min, twice in 90% ethanol for 20 min and twice in 100% ethanol for 5 min (all at RT). Juveniles were covered with 200 μl hexamethyldisilazane (Sigma-Aldrich) and after 5 min this was removed and 200 μl of fresh hexamethyldisilazane was added and allowed to evaporate overnight (~16 h, RT). Juveniles were then transferred onto stubs and sputter coated for 5 min using a Polaron E5100 Series II before viewing under a FEI Quanta 200 scanning electron microscope. Image J software was used to measure the length of ten spines within the first three rings surrounding the oral suckers of 2–3 juveniles at each week of culture following excystment.
7
Scanning Electron Microscopy Analysis of Satsuma Mandarin Leaves
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Scanning election microscopy (S-3500N, Hitachi Corporation, Japan) analyses were carried out to observe initial symptom development, cell division, lignin deposition and development of lamellated cell barrier in satsuma mandarin leaves at an accelerating voltage of 20 kV. Inoculated leaves were harvested, cut approximately into 0.5 cm × 0.5 cm pieces and fixed in Karnovsky’s fixative solution [2% gluteraldehyde (Sigma-Aldrich, Germany) and 2.5% paraformaldehyde (Sigma-Aldrich, Germany) in 0.05 M sodium cacodylate (prepared from cacodylic acid, Sigma-Aldrich, Germany) buffer pH 7.2 to 7.4] for 2 h at room temperature. And then the samples were washed with 0.05 M sodium cacodylate buffer two times for 20 min each and allowed by fixation with second fixative, 1% osmium tetroxide (Fluka Analytical, USA) for 2 h. They were dehydrated using an ethanol series from 10, 20, 30, 40, 50, 60, 70, 80, 90, 95 percent for 15 min each and in 100% ethanol three times for 15 min each. The critical dehydration was finally done by using chemical dehydrant HMDS (hexamethyldisilazane, Merck) or HCP-2 critical point dryer (Hitachi Corporation, Japan) according to instructions of the manufacturer. The specimens were mounted on aluminum stubs. If required, specimens were sputter coated with evaporated platinum for 50 s in vacuum using E-1030 Ion sputter coater unit (Hitachi Corporation, Japan).
8
Fabrication and Characterization of Neurotrophin-Loaded PLGA-PDLLA Scaffolds
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PLGA (LA:GA = 50:50) and PDLLA with molecular weight of 100 kDa (as indicated by their inherent viscosity 0.6–0.8 dL/g) were purchased from Lakeshore Biomaterials, USA. Chloroform was supplied by Uni Chem Co., Korea. The human β-NGF with Enzyme Linked Immunosorbent Assay (ELISA) Kit, human GDNF with ELISA Kit were purchased from Peprotech Inc. and R&D Systems, Inc., respectively. Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), and trypsin/EDTA were purchased from Invitrogen, Inc., USA. Mouse anti-neurofilament (M + H) primary antibody, goat anti-mouse IgG secondary antibody (Alexa Fluor® 594), phalloidin (Alexa Fluor® 488), and DAPI (4’, 6-diamidino-2-phenylindole) were purchased from Life Technologies. Triton X-100, Span-80, phosphate buffered saline (PBS) tablets, heparin, bovine serum albumin (BSA), paraformaldehyde, glutaraldehyde, sodium cacodylate, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide), and sucrose were Sigma-Aldrich products. Other chemicals were used as received.
9
Enzymatic Analysis of Cellulose Oligomers
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Cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose were from Megazyme (Bray, Ireland). Sodium cacodylate, manganese (II) chloride tetrahydrate, uridine diphosphate galactose (UDP-Gal), fluorescein isothiocyanate (isomer I) and galactosyl transferase from bovine milk were from Sigma. AlexaFluor488 C5-aminooxyacetamide, and bis(triethylammonium) salt were from Invitrogen (Nærum, Denmark). 2-(aminooxy)-1-ethanaminium dichloride was from ABCR GmbH (Karlsruhe, Germany). 2,5-Dihydroxy-benzoic acid was from Bruker Daltonics (Bremen, Germany). The LPMO used in this study, from Neurospora crassa (NcLPMO9A), and Cellobiose dehydrogenase from Myrococcum thermophilum (MtCDH) were produced and purified according to Petrovic et al. 201926 (link) and Flitsch et al. 201928 (link), respectively. The endocellulase Cel5A from Hypocrea jecorina was produced according to Saloheimo et al. 198841 (link) and the exocelluase Cel6B from Thermobifida fusca was produced according to Vuong and Wilson 200942 (link).
10
Biofilm Imaging After Statin Treatment
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