Twenty-nine male Wistar rats, weighing 200–250 g and aged seven weeks, were acquired from Charles River (France). Sigma-Aldrich® (Saint Louis, MO, USA) provided DMH, which was prepared immediately before its use in a saline solution containing 0.9% of NaCl (B. Braun, Melsungen, Germany) and 1 mM of ethylenediaminetetraacetic acid (EDTA; E5134, Sigma-Aldrich®, Saint Louis, Missouri, USA) [10 (link)]. During the study, the rats were kept in controlled conditions of temperature (20 ± 2 °C), relative humidity (50 ± 10%), and light-dark cycle (12:12-h). The rats were housed in groups of three to five animals per cage (1500UEurostandard Type IV S cages, Tecniplast, Buguggiate, Italy). Food (Diet Standard 4RF21®, Mucedola, Italy) and tap water were provided ad libitum throughout the study. Corncob was used as bedding and was changed weekly. Polyvinyl chloride tubes were placed into the cages as environmental enrichment for the animals. The viruses and bacteria specified in the Supplementary Materials (Table S1 ), as well as endo and ectoparasites, were not found in the rats.
E 5134
Manufactured by Merck Group
Sourced in United States
E-5134 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of E-5134 is to perform specific tasks required in a laboratory setting. No further details or interpretation of the intended use are provided.
Automatically generated - may contain errors
Lab products found in correlation
Link 48, by Agilent Technologies (2 mentions)
H324 500, by Thermo Fisher Scientific (2 mentions)
Aperio cs2, by Leica camera (2 mentions)
E046201, by Agilent Technologies (2 mentions)
Imagescope software, by Leica camera (2 mentions)
Wistar rat, by Charles River Laboratories (1 mentions)
Sodium chloride (nacl), by B. Braun (1 mentions)
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
B1000, by Biocolor (1 mentions)
Papain, by Merck Group (1 mentions)
Blyscan, by Biocolor (1 mentions)
Catalase from bovine liver, by Merck Group (1 mentions)
E8875, by Merck Group (1 mentions)
17 estradiol, by Merck Group (1 mentions)
Infinite 200 pro, by Tecan (1 mentions)
Pepsin p7000, by Merck Group (1 mentions)
Naringenin n5893, by Merck Group (1 mentions)
Millipore milli q system, by Merck Group (1 mentions)
Methanol, by Merck Group (1 mentions)
Hexane, by Merck Group (1 mentions)
12 protocols using e 5134
1
Wistar Rat Protocol for DMH-Induced Colorectal Cancer
2
Measurement of Sulfated GAG and DNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured samples were washed with Dulbecco’s phosphate-buffered saline (DPBS; DSBN200, DS-Pharma, Osaka, Japan) and digested at 65°C with 0.5 mg/ml papain (P3125, Sigma-Aldrich) dissolved overnight in a 0.2 M sodium phosphate buffer solution supplemented with 8 mg/ml sodium acetate (#192–01075 Wako Pure Chemical), 4 mg/ml ethylenediaminetetraacetic acid sodium salt dihydrate (E-5134, Sigma-Aldrich) and 0.8 mg/ml L-cysteine (C-7880, Sigma-Aldrich). Sulfated GAG content was measured with a 1,9-dimethyl-methylene blue (DMMB) assay (Blyscan; B1000, Biocolor, Belfast, UK) with chondroitin-6-sulfate (B1000, Biocolor) as the standard, using a spectrophotometer to measure absorbance at 656 nm. DNA quantity was determined using a PicoGreen assay (P11496, Thermo Fisher Scientific) with a spectrophotometer in fluorescence mode with excitation at 480 nm and emission at 520 nm. Each colorimetric measurement was done for three wells in 96 well plates for each group of samples from five donors. NTP concentrations of 0.1 and 1.0 mNU/ml were used.
3
Quantifying Ascorbate Oxidation in the Presence of Oxidants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
O2 consumption measures were done using a Clark electrode system; an ESA BioStat Multi-Electrode System (ESA Products, Dionex Corp, Chelmsford, MA) in conjunction with a YSI Oxygen Probe (5331) and glass reaction chamber vials in a YSI bath assembly (5301) (Yellow Springs Instruments, Yellow Springs, OH). Both AscH− and FMX were added to 100 mM KPO4 buffer, without any prior dilutions, inside of the Clark electrode system. Before adding FMX, AscH− was allowed to autoxidize for a minimum of 5 min to gather background oxidation rates. Ethylenedinitrilotetraacetic acid (EDTA; Sigma-Aldrich, E−5134), desferrioxamine mesylate salt (DFO; Sigma-Aldrich, D9533), and catalase from bovine liver (Sigma-Aldrich, C1345) were diluted in double-distilled H2O before addition to the electrode system and were added after 5 min of AscH− and FMX co-incubation.
4
Assay for 3β-HSD and 17β-HSD Enzyme Activities
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Enzyme activity of 3ß HSD and 17ß HSD was measured using a pre-established method30 (link). Freshly collected ovary and adrenal tissues were homogenized using a homogenizing solution (1 ml/100 mg of tissue) containing glycerol, 5 mM potassium phosphate (60350 Sigma), 1 mM EDTA (E5134 Sigma) and distilled water and centrifuged at 10,000 × g for 30 mins at 4 °C. The supernatant was collected and used for both the enzyme activity measurements. For 3ß HSD activity, 100 µM tetrasodium pyrophosphate (P8010 Aldrich), 1 ml of 0.5 mM NAD (93205 Sigma) and 30 µg 17ß-estradiol (E8875, Sigma) were added along with 100 µl of the sample. For 17ß HSD activity, 440 µM tetrasodium pyrophosphate, 1.35 µM NAD, 0.3 µM 17ß-estradiol and 5% Bovine Serum Albumin (BSA) (A2153 Sigma) were added along with 100 µl of the sample. The kinetic assay was measured by using a multireader (Tecan Infinite 200 PRO, Switzerland) for 3 minutes (15 seconds interval) at 340 nm (1U = change in absorbance of 0.001/ min) and expressed as enzyme activity/ min/ mg protein.
5
Analytical Determination of Bioactive Compounds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
β-glucuronidase (G0751), ethyl acetate, hexane, isopropyl alcohol, methanol, Na2-ethylenediaminetetraacetic acid (E5134) and pepsin (P7000) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). L-Ascorbic acid and NaOH were obtained from Avantor Performance Materials Poland S.A. (Gliwice, Poland). HCl and formic acid were purchased from Merck KGaA (Darmstadt, Germany). Naringenin (N5893) was obtained from Sigma-Aldrich Co. (St. Louis, MO, USA). Millipore Milli-Q system (Millipore Co., Millford, MA, USA) was used to produce ultrapure water.
6
White Blood Cell Extraction Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Blood samples were drawn from the cubital vein into EDTA tubes and refrigerated at 4 °C until processed. For white blood cell (WBC) extraction, up to 4 mL of each blood sample were processed in Falcon tubes containing 6 mL of lysis solution for red blood cells (RBC) (RBC lysis solution: 1 mM EDTA solution obtained by a 0.5 M EDTA solution at pH 8.0, prepared with EDTA disodium salt dihydrate from Sigma E-5134, and stored at room temperature). Tubes were mixed by inversion 2 times, incubated for 2 min at room temperature, mixed once more time by inversion and centrifuged at 2000 g (3161 rpm) for 20 min at room temperature. After centrifugation, supernatants were removed, leaving the WBC pellets at the bottom of the tubes. This procedure was repeated at least 3 or 4 times. Cells were suspended in 1 mL of 1 M NaCl Solution, aliquoted and frozen at −20 °C until use.
7
Ototoxic Damage Induction Protocol
8
Harvesting and Isolating Tumor-Infiltrating DCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The subcutaneous LLC tumor model was employed by injecting 2.5 × 105 LL/2 LLC cells in 50 μl DPBS subcutaneously using a 25-g needle in the mouse left lower abdominal wall and harvesting tumors at day 20. Mice were euthanized using carbon dioxide according to current American Veterinary Medical Association guidelines. Tumors were extracted in a sterile fashion, weighed, and minced with scissors and digested for 1 hour in a digestion buffer solution containing DMEM, 0.2% collagenase from Clostridium histolyticum (Sigma-Aldrich, C2799), and 15 μg/ml DNAse I (Sigma-Aldrich, D4513). Single-cell suspensions were obtained by filtering through a 40-μm cell strainer (Fisherbrand, 22363547). Magnetic separation of DCs (CD11c+ cells) was performed according to Miltenyi Biotec manufacturer instructions. Briefly, cells were magnetically labeled with CD11c MicroBeads (120-000-322), loaded onto a MACS MS column (130-042-20), and placed in the magnetic field of a magnetic separator (MiniMACS 130-042-102). The unlabeled CD11c− cells were depleted by running a MACS buffer solution (DPBS, 0.5% BSA [Gemini Bio-Products 700-100P], and 2 mM EDTA [Sigma-Aldrich, E5134]) through the column. Finally, the MACS MS column was removed from the magnetic field and flushed out using a MACS buffer solution and a plunger to collect the magnetically labeled CD11c+ cells.
9
Automated Immunohistochemistry for TTF1 in FFPE
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemistry (IHC) was performed on formalin-fixed paraffin-embedded (FFPE) patient sections with an automated staining system (Dako Link48) using TTF1 primary antibodies (ZSGB-Bio Cat: ZM-0270). After deparaffinization and rehydration in xylene and ethanol, antigen retrieval was performed in 1× EDTA retrieval solution (pH 8.0) (E-5134, Sigma) with heating. Inactivation of endogenous peroxidase was performed by adding enough drops of 3% hydrogen peroxide (H324-500, Fisher) to cover the whole section for 10 min and followed by primary antibody incubation at recommended dilution. After 60 min incubation at 37 °C, slides were washed twice with PBS, incubated with goat-anti-rabbit antibody (E046201, Dako) and then developed with DAB substrate (k3468, Dako) for 5–8 min depending on the antibodies. Slides were counterstained with hematoxylin (CTS-1090, Biotechnologies) and were scanned with brightfield pathology microscope at 20× magnification (Aperio CS2, Leica) and processed by ImageScope software (Leica). Information on all other chemicals and reagents used in the article can be found in Supplementary S-2 .
10
Slice-in-Place Technique for Intact Skull Sectioning
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In order to maintain placement of individual electrode fibers, the skull was extracted with the implant intact and sectioned using a slice-in-place technique (Patel et al., 2020 (link)). Briefly, animals were sedated with Fatal Plus (Vortech Pharmaceuticals, V.P.L. 9373) and transcardially perfused with 0.1 M phosphate buffered saline (PBS) followed by 4% paraformaldehyde (ChemCruz, SC-281692). The animal was then decapitated, and the skull was stripped of skin, ensuring the implant remains cemented to the skull. The skull was then soaked in 0.25 M ethylenediaminetetraacetic acid (Sigma, E5134) in 0.1 M PBS solution at 4 °C with low agitation. The solution was changed every 24 hours for 14 days. The softened skull was then transferred into a 30% sucrose (Sigma, S0389) solution with 0.02% sodium azide (Sigma, S2002) for 48 hours or until sectioning. Before sectioning, skulls were immersed in OCT (ThermoFisher, 4585) and put under a −0.06 MPa vacuum. Skulls were frozen and mounted on a cryostat (ThermoFisher, HM 525NX). 300 μm horizontal sections were taken at −19 °C and stored in 0.1 M PBS with 0.02% sodium azide.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!