The hdECM was prepared as described previously16 (link),17 (link). Briefly, heart tissue from a 6-month-old Korean domestic pig was purchased from a livestock product market with supplier approval. We dissected the left ventricle from the complete porcine heart and cut it into small pieces. The small pieces of heart tissue were soaked in 1% sodium dodecyl sulfate (Affymetrix, CA) solution for 48 h followed by treatment with 1% Triton X-100 solution in PBS (Biosesang, Korea) for 1 h. Next, the decellularized tissues were dipped in PBS for 3 days to remove the residual detergent. Subsequently, the decellularized heart tissues were lyophilized, pulverized in liquid nitrogen and digested in 10 mL of 0.5 M acetic acid solution (Merck Millipore, Billerica, MA) at a final concentration of 3.3 w/v% (330 mg of hdECM powder) with 33 mg of pepsin powder. The digested hdECM solution was filtered through a 40 µm pore mesh, aliquoted in 1 mL, and stored at -20 °C for further experiments. Before the cardiac patches were manufactured, the hdECM solution was adjusted to a neutral pH of 7.4 by adding 10 N NaOH solution while keeping the conical tube in an ice bucket to avoid gelation of hdECM.
Phosphate buffered saline (pbs)
Manufactured by Biosesang
Sourced in United States
PBS (Phosphate-Buffered Saline) is a commonly used buffer solution in various laboratory applications. It is an isotonic solution that maintains a physiological pH and osmolarity, making it suitable for the preservation and manipulation of biological samples. PBS is a versatile and widely-used buffer in procedures such as cell culture, immunoassays, and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Isopropanol, by Merck Group (3 mentions)
Sodium dodecyl sulfate (sds), by Biosesang (2 mentions)
Tween 20, by Bio-Rad (2 mentions)
P gsk3β, by Cell Signaling Technology (2 mentions)
Methyl thiazolyl tetrazolium (mtt), by Biosesang (2 mentions)
Tris buffered saline (tbs), by Biosesang (2 mentions)
L dopa, by Merck Group (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
β catenin, by Cell Signaling Technology (2 mentions)
P β catenin, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Sodium hydroxide (naoh), by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
Ripa buffer, by Biosesang (2 mentions)
Dimethyl sulfoxide (dmso), by Biosesang (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
22 protocols using phosphate buffered saline (pbs)
1
Porcine Decellularized Cardiac ECM Preparation
2
Amyloid-β Quantification Assay Protocol
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ADHP (10-acetyl 3,7-dihydroxyphenoxazine, Amplex Red) was purchased at Invitrogen (Carlsbad, CA, USA). Hydrogen peroxide was obtained from a Quanta Red Enhanced Chemifluorescent HRP Substrate kit (Thermo Scientific, Waltham, MA, USA). Resorufin (424455) and phosphate buffered saline (PBS) was purchased from Sigma Aldrich (St. Louis, MO, USA) and Welgene (Gyeongsangbuk-do, Korea) respectively. PBS of various pH was purchased from the Biosesang (Seongnam-si, Gyeonggi-do, Korea). Biotin-coated plate and HRP-conjugated streptavidin were purchased from Thermo Scientific. Human Aβ42 ELISA kits were purchased from Invitrogen. Pooled Human Plasma Apheresis Derived was purchased from Innovative Research (Novi, MI, USA).
3
Investigating hGH Ubiquitination in Blood
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hGH-His was constructed by removing the stop codon from the aforementioned pET-28a-hGH using site-directed mutagenesis for PCR. The protein was purified by the same purification method as described above. In addition, then, to investigate whether hGH is ubiquitinated in the blood stream, 300 μL mouse whole blood was cleared for 1 h at 4 °C using 300 μL protein A/G Agarose (sc-2003, Santa Cruz), then centrifuged twice at 3000 rpm for 1 min to obtain supernatants. An amount of 100 ng of purified hGH-His was incubated for 1 h at 37 °C with MG132 (Sigma-Aldrich) (5 μg/mL) in 1.3 mL mouse whole blood and pull-down assay was performed using 30 μL Ni-NTA beads (Qiagen, Hilden, Germany). Then, the precipitant for hGH-His was washed five times with phosphate-buffered saline (PBS, Biosesang, Seongnam-si, Korea).
4
Multicolor Immunofluorescence Staining Protocol
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Paraffin sections were deparaffinized and rehydrated. Antigens were retrieved with citrate buffer, pH 6.0 (Dako, Agilent technologies, Glostrup, Denmark). Sections were permeabilized with 0.2% Triton x-100 (Promega, Fitchburg, WI, USA) in PBS (Biosesang, Bundang, Korea), washed with PBS, blocked for 1 h with 3% bovine serum albumin (BSA) (Sigma Aldrich), 3% donkey serum (Sigma Aldrich) in PBS with 0.05% Tween 20 (Amresco, Solon, OH, USA). Sections were washed 3 times with PBS and diluted primary antibodies were added for overnight at 4 °C. Primary antibodies were as follows: mouse anti-human CD47 (Thermo Fisher Scientific), rabbit anti-human CD68 (Cell signaling technology, Danvers, MA, USA), goat anti-human CD14 (Thermo Fisher Scientific). After washing with PBS, diluted secondary antibodies were added for 1 hour at room temperature. Secondary antibodies were as follows: donkey anti-mouse IgG Alexa Fluor 488, donkey anti-rabbit IgG Alexa Fluor 594, and donkey anti-goat IgG Alexa Fluor 647 (all from Thermo Fisher Scientific). After washing, sections were mounted with mounting solution which includes DAPI (Merck).
5
Bacterial Cell Fixation and Permeabilization
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The bacterial cell fixation was performed by modified fixation method [25 (link)]. Cultured bacterial cells were harvested by centrifugation at 12,000 rpm for 1 min and discarded the supernatant. Cell pellets were washed with PBS (Biosesang Inc. Co., Seongnam, Korea, Cat. No. P2004) three times. For the permeabilization of the cell, ice-cold 4% paraformaldehyde in PBS was applied for 3–12 h at 4 °C. For the fixation of bacterial cells, the permeabilized cells were resuspended with ice-cold 50% EtOH. Next, 10 μL of fixed cell suspension was dropped on the gelatin coated-slide glass and dried under air flow. The slide glass was soaked in the solution of 50% EtOH for 3 min, 80% EtOH for 3 min, and 95% EtOH for 3 min. Finally, the slide glass was air dried.
6
Isolation and Culture of Mouse ADSCs
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ADSCs were obtained from subcutaneous adipose tissue of C57BL/6 mice and isolated using previously established methods15 (link)
. Fat tissues were washed by PBS (Biosesang, Seongnam, Korea) and then finely minced and digested with collagenase type I (Sigma-Aldrich, Albuch, Germany) in an incubator for 40 minutes at 37°C. Then, the cell suspension was mixed with low-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Seoul, Korea) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% penicillin/streptomycin (Invitrogen, Waltham, Massachusetts, USA) and centrifuged at 420 g for 5 min. Subsequently, the cell pellet was washed twice with PBS and re-suspended in the same medium. Cells were plated and incubated at 37°C in 5% CO2. Adherent cells were maintained in culture for four passages. At passage four, ADSCs presented spindle-shaped marphology, and when these cells were approximately 90% confluent, they were digested with 0.25% trypsin/EDTA (Gibco, Gaithersburg, MD, USA) at 37°C, washed with PBS, and pelleted by centrifugation. The number of cells was adjusted to 1.5 × 105 (link)
/mL in 0.1 mL PBS for injection.
. Fat tissues were washed by PBS (Biosesang, Seongnam, Korea) and then finely minced and digested with collagenase type I (Sigma-Aldrich, Albuch, Germany) in an incubator for 40 minutes at 37°C. Then, the cell suspension was mixed with low-glucose Dulbecco’s modified Eagle’s medium (Invitrogen, Seoul, Korea) supplemented with 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA) and 1% penicillin/streptomycin (Invitrogen, Waltham, Massachusetts, USA) and centrifuged at 420 g for 5 min. Subsequently, the cell pellet was washed twice with PBS and re-suspended in the same medium. Cells were plated and incubated at 37°C in 5% CO2. Adherent cells were maintained in culture for four passages. At passage four, ADSCs presented spindle-shaped marphology, and when these cells were approximately 90% confluent, they were digested with 0.25% trypsin/EDTA (Gibco, Gaithersburg, MD, USA) at 37°C, washed with PBS, and pelleted by centrifugation. The number of cells was adjusted to 1.5 × 105 (link)
/mL in 0.1 mL PBS for injection.
7
Melanogenesis Regulation via Signaling Pathways
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Syringetin (CAS 4423-37-4) was purchased from Extrasynthese (Genay Cedex, France), while α-melanocyte stimulating hormone (α-MSH), protease/phosphatase inhibitor cocktail, sodium hydroxide (NaOH), and L-DOPA were obtained from Sigma-Aldrich (St. Louis, MO, USA). MTT, DMSO, PBS, TBS, SDS, RIPA buffer, and the ECL kit were purchased from Biosesang (Seongnam, Gyeonggi-do, Korea), while DMEM, penicillin–streptomycin, BCA protein assay kits, and 0.5% trypsin-ethylenediaminetetraacetic acid (10×) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). For Western blots, the primary antibodies against tyrosinase, TRP-1, TRP-2, and MITF were purchased from Santa Cruz Biotechnology (Dallas, TX, USA), while the other antibodies, against p-ERK, ERK, p-p38, p38, p-JNK, JNK, p-PKA, PKA, p-AKT, AKT, p-GSK-3β, GSK-3β, p-β-catenin, β-catenin, β-actin, anti-rabbit, and secondary antibodies, were purchased from Cell Signaling Technology (Danvers, MA, USA). Skimmed milk was purchased from BD Difco (Sparks, MD, USA), fetal bovine serum (FBS) from Merck Millipore (Burling, USA), and Tween 20 and 2 × Laemmli sample buffer were obtained from Bio-rad (Hercules, CA, USA).
8
Histological Assessment of Ear Tissue
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Ear tissues were fixed in 10% formalin solution (Sigma, Mo, USA) for 24 h. Tissues were washed with PBS (Biosesang, Seongnam, Korea), embedded in paraffin (Leica Biosystems, Wetzlar, Germany) and sectioned 4 μm thickness using microtome (HistoCore BIOCUT; Leica Biosystems, Germany). Stained images were taken using light microscope (Olympus, Tokyo, Japan). Mast cells or eosinophils were counted in 3 random high-power field (HPF) each mouse at 400× magnification. H&E staining was performed according to previously described34 (link). Epidermis and dermis thickness were measured using imageJ software program. Toluidine blue staining was used to count infiltrated mast cells. Briefly, hydrated tissue sections were stained with 0.1% Toluidine Blue O in 1% sodium chloride solution (pH 2; Sigma, USA) for 1 min. After staining, sections were washed briefly in PBS, and dehydration 3 times with 95% to 100% ethanol, and then sealed using mounting medium35 (link). Sirius Red staining was used to count the number of infiltrated eosinophil cells36 (link). Reagent preparation and staining protocol were according to previously as described37 (link).
9
Immunohistochemical Analysis of Dopaminergic Neurons
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In an in vivo experiment, after the last behavior tests were completed, mice were sacrificed, and brains were quickly harvested, fixed in 4% paraformaldehyde for 24 h, and kept in 30% sucrose solution at 4 °C until they are sectioned. The brain samples were embedded in an optimal cutting temperature compound (Sakura Finetek, Torrance, CA, USA) for 3 h and frozen sections were cut to a thickness of 20 µm using a cryostat (Thermo Fisher Scientific, Waltham, MA, USA). Frozen sections were blocked with 1% bovine serum albumin (BSA; VWR Life Science, Radnor, PA, USA) in 0.01 M PBS for 1 h at room temperature. After blocking, samples were incubated with rabbit anti-TH primary antibody (1:1000; Novus, St. Charles, MO, USA), and CD-68 (1:50; Thermo Fisher Scientific, Waltham, MA, USA) overnight at room temperature. The samples were washed with PBS (Biosesang, Seoul, Korea) and incubated with goat anti-mouse and goat anti-rabbit IgG (H+L) antibodies, Alexa Fluor 568 (1:2000; Invitrogen, Waltham, MA, USA) for 1.5 h at room temperature. After washing with 0.01M PBS, samples were mounted with Fluoroshield with DAPI (Sigma-Aldrich, St. Louis, MO, USA). All the stained samples were detected by a fluorescence microscope (Olympus, Tokyo, Japan).
10
Pimozide Treatment in TRAMP Mice
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The experimental protocol for animal handling was in accordance with the National Institute of Health (NIH) guidelines and approved by the Institutional Animal Care and Use Committee of Seoul National University (Protocol Number: SNU-181128-1). Male TRAMP mice expressing the SV40 large T-antigen under control of the prostate-specific rat probasin promoter were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and housed in the Animal Experiment Facility, College of Veterinary Medicine, Seoul National University. Mice were kept on a 12-hr light/dark cycle with ad libitum access to food and water. Pimozide was suspended in 10% DMSO, 10% Tween80 (Sigma), and 80% PBS (Biosesang, Seongnam, South Korea). The mice were randomly assigned to control and treatment groups (n = 5 for control and pimozide 10 mg/kg groups, and n = 3 for pimozide 5 mg/kg group). Pimozide at 5 and 10 mg/kg/5 times per week was administered by intraperitoneal injection to TRAMP males beginning at 12 weeks of age and was continued until the animals were 24 weeks old at which time the experiment was terminated. Body weight was measured weekly.
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