A small hole was pricked into the cornea of the median ocellus to allow the insertion of a 10 µl-syringe (World Precision Instrument), which was used to inject 200 nl of each drug solution21 (link). Drugs were injected into the brain of immobilized bees along the median ocellar nerve21 (link). This method ensures that drugs migrate through the ocellar tract and distribute within the bee brain in a fast (less than 5 min) and homogenous way76 (link). Thirty min before the experiment, we injected: Epinastine hydrochloride, an OA receptor antagonist46 (link), cis-(Z)-flupentixol dihydrochloride, a DA receptor antagonist47 (link); or PBS (control). Epinastine and flupentixol were obtained from Sigma-Aldrich (Saint-Quentin Fallavier, France). PBS was obtained from EUROMEDEX (Strasbourg, France). Injection time was chosen based on experiments, which showed that the effects of aminergic blockers reach a stable level approximately 30 min after drug application35 (link),77 (link)–79 (link). One mg of each drug was diluted in 1 ml PBS. Final concentrations obtained were 3.5 mM of Epinastine and 1.94 mM of flupentixol. To test for dose-response effects, we prepared for each drug an additional dilution of 1:10000. In all cases, aliquots were kept in −20 °C until use. Each aliquot was used for one whole week and kept during this time in 4 °C.
Phosphate buffered saline (pbs)
Manufactured by Euromedex
Sourced in France
PBS (Phosphate-Buffered Saline) is a commonly used buffer solution in various laboratory applications. It is a balanced salt solution that maintains a stable pH and osmolarity, providing a physiologically compatible environment for biological samples. The core function of PBS is to maintain the viability and integrity of cells, tissues, or other biological materials during experimentation or storage.
Automatically generated - may contain errors
Lab products found in correlation
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Flupentixol, by Merck Group (1 mentions)
Epinastine, by Merck Group (1 mentions)
10 l syringe, by World Precision Instruments (1 mentions)
Fraction 5, by Roche (1 mentions)
Tla 100, by Beckman Coulter (1 mentions)
Complete edta free, by Roche (1 mentions)
Protease inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Bovine serum albumin (bsa), by Euromedex (1 mentions)
Collagenase 1, by Thermo Fisher Scientific (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Santa Cruz Biotechnology (1 mentions)
Hepes buffer, by Thermo Fisher Scientific (1 mentions)
Maxymum recovery, by Corning (1 mentions)
Peg nhs, by Merck Group (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
5 protocols using phosphate buffered saline (pbs)
1
Intracranial Injection of Aminergic Antagonists in Bees
2
Fibroblast Lectin Staining Protocol
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Patients and control fibroblasts (80–90% confluence) were cultured on coverslips in six well plates in DMEM supplemented with 10% FBS, under 5% CO2 atmosphere. For lectin staining, coverslips were washed 3 times with PBS (+/+) (Euromedex, Souffelweyersheim, France) and mounted in a wet chamber. PNA-Cy5 or VVL-Fluorescein lectins (Vector laboratories, California, USA) were diluted at 2 μg/ml in PBS + 0.1% of BSA (Fraction V, Roche Diagnostics, Rotkreuz, Switzerland), applied on coverslips and incubated for 1 h at room temperature protected from light. The coverslips were then washed three times and fixed with 4% paraformaldehyde in PBS pH 7.3, for 30 min at room temperature. Nuclei were stained with DAPI (PBS) and coverslips were then mounted on glass slides with Mowiol.
3
Colicin Quantification by Western Blot
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50 ml cultures of wild-type or mutant E. coli strains were grown in LB to OD600 = 0.4, at 37°C, then 1 mg of colicin E-ImmE complex was added to the cultures for 45 min. The cells were harvested, washed three-time, resuspended in phosphate buffer pH 7.4 (PBS, Euromedex), and treated with proteinase K at 100 µg/ml for 1 hour at 37°C, to hydrolyse residual colicin molecules bound to the cell surface. After four additional washings the colicin- and proteinase K-treated cells were resuspended in 2 ml PBS buffer with a protease inhibitor complex (Complete EDTA-free; Roche) and then disrupted by sonication. Ribosomes of the soluble fraction were eliminated by micro-ultracentrifugation at 100 S (Beckman rotor TLA 100.2). Proteins of the S100 cytoplasmic fraction (extract) were separated on 15% SDS-PAGE and analysed by Western blotting with anti-colicin E2-ImmE2 (or E3-ImmE3 or E7-ImmE7) antiserum and detected by ECL chemiluminescence (BIO-RAD, Immun-Star) associated with a CCD camera (BIO-RAD ChemiDoc XR System+). When necessary, ECL detected proteins were quantified by ImageLab software (BIO-RAD).
4
Isolation and Culture of Primary Podocytes
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Freshly isolated renal cortexes from Stat5lox/lox and Nphs2-Cre;Stat5lox/lox mice were minced and digested by collagenase I (1 mg/ml; Invitrogen) in RPMI 1640 (Invitrogen) for 2 minutes at 37 °C. Next, collagenase I was inactivated by addition of RPMI 1640 plus 10% FBS (Hyclone). Digested tissues were passed through a 100-μm cell strainer. The strainer was rinsed with 15 ml of PBS (Euromedex) containing 0.5% BSA (Euromedex). The flow-through was then passed through a 40-μm cell strainer (BD Falcon). Glomeruli, adherent to the 40-μm cell strainer, were removed with PBS-0.5% BSA injected under pressure, and finally washed twice in PBS. Freshly isolated glomeruli purity was analyzed by light microscopy and then plated in 6-well plates in RPMI-1640 containing 10% FBS, 2% HEPES buffer (Gibco), and 1% penicillin/streptomycin (Gibco). Two days after seeding, glomeruli became adherent to the dish, and podocytes spread out from glomeruli. Primary podocytes were then lysed in RIPA buffer (Santa Cruz) with orthovanadate, PMSF, protease inhibitor cocktail (Santa Cruz) and NaF for protein extraction.
5
Fabrication of Hydrogel Discs and Drops
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Poly(L-lysine) dendrimers of third generation (DGL), (molecular weight of 22000 g/mol, Colcom, France) and Poly(ethylene glycol) (PEG)-bis(N-succinimidyl succinate) (PEG-NHS, 2000 g/mol, Sigma Aldrich) were solubilized at 400 mg/mL in phosphate buffered saline (PBS, Euromedex) and DMSO (Sigma Aldrich) respectively before use. Stock solutions of PEG-NHS in DMSO and DGL in PBS (400 mg/mL) were added to the adjusted volume of PBS to obtain the desired concentrations (i.e; 1.6/25, 2/25 and 2/37 mM DGL/PEG) in 2 ml conical tubes (Maxymum Recovery, Axygen) followed by vigorous homogenization. To form cylinders, 400 µL of hydrogel precursors were allowed to crosslink inside the tubes to then be retrieved and sectioned using a vibratome (7550 Integraslice) at a 50 Hz frequency, 1 µm amplitude and a slow blade speed of 0.10 to 0.15 mm/s to obtain hydrogel discs of 2 or 3 mm high and 9.1 mm diameter. To form drops, 90 µL of the hydrogel precursor mix were swiftly deposited onto a PTFE plate, immediately after homogenization. Hydrogels were allowed to crosslink for 10 min in wet chambers, detached from the hydrophobic surface and immediately used for subsequent experiments without any washing.
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