Cytokines (IL-37, IL-19, CXCL1, CXCL2 and CXCL13) were purchased from Cloud-Clone Corp (Wuhan, China), and L-dopa and sodium deoxycholate from Solarbio (Beijing, China). TritonX-100 was purchased from Sigma-Aldrich. Cell Counting Kit-8 (CCK8) and 4% neutral paraformaldehyde were from Biosharp (Hefei, China). Fetal bovine serum (FBS) from Meisen (Zhejiang, China). Dulbecco’s modified Eagle medium (DMEM), penicillin/streptomycin/amphotericin B, penicillin-streptomycin (P/S), and non-essential amino acids (NEAA) were from Gibco (Maryland, USA). Primary antibodies specific for GAPDH (#AP0066, Bioworld), TYR (BS1484, Bioworld), MITF (STJ94134, St. John’s Laboratory), TYRP1 (ab235447, Abcam), DCT (NBP1-56058, Novusbio), PMEL17(#H1219, Santa Cruz and bs-17478R, Bioss) and MLANA (bs-0051R, Bioss and ET1610-47, HUABIO) were purchased as indicated.
Neutral paraformaldehyde
Manufactured by Biosharp
Sourced in United States, China
4% neutral paraformaldehyde is a fixative used in various laboratory applications. It is a 4% solution of paraformaldehyde, a polymer of formaldehyde, in a neutral pH buffer. This product is commonly used for the preservation and fixation of biological samples, such as cells and tissues, prior to further analysis or processing.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Merck Group (3 mentions)
Sodium deoxycholate, by Solarbio (2 mentions)
Ab235447, by Abcam (2 mentions)
Nbp1 56058, by Novus Biologicals (2 mentions)
L dopa, by Solarbio (2 mentions)
H e staining, by Solarbio (2 mentions)
Fv1000, by Olympus (2 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Cxcl1, by Cloud-Clone (1 mentions)
Cxcl2, by Cloud-Clone (1 mentions)
Penicillin streptomycin p s, by Thermo Fisher Scientific (1 mentions)
Cell counting kit 8 cck 8, by Biosharp (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Tunel apoptosis assay kit, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Medium 254, by Thermo Fisher Scientific (1 mentions)
2 7 dichlorofluorescein diacetate dcfh da, by Beyotime (1 mentions)
Primary antibodies for β actin, by Cell Signaling Technology (1 mentions)
Rab27a, by Cell Signaling Technology (1 mentions)
7 protocols using neutral paraformaldehyde
1
Cytokine-Mediated Melanogenesis Regulation
2
Histological Evaluation of Wound Samples
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To conduct histological evaluation, the harvested samples were fixed overnight in 4% neutral paraformaldehyde (Biosharp, USA), dehydrated in gradient ethanol, transparent in xylene, and finally embedded in paraffin and cut into 5–6 µ m thick sections. The sections near the center of the wound were used for further testing. H&E staining (Solarbio, China) was performed to visualize the structure of wounds and scars. Masson staining (Baso, China) was performed to visualize the collagen fibers according to the manufacturer’s instructions.
3
Transwell Assay for Cell Migration
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Transwell assays were also used to analyze the migration ability of cells. HFFs or HUVECs were seeded at 1.5×104 cells per well into the upper chamber of a Transwell (Corning, USA). Cells were divided into the blank group and the ApoEVs-AT group. In the blank group, 500 μL PBS was added to the lower chamber. In the ApoEVs-AT group, 500 μL PBS containing 25 μg ApoEVs-AT was added to the lower chamber as a chemoattractant. After culturing for 12 h, nonmigratory cells in the upper chamber were removed. The migrated cells were fixed with 4% neutral paraformaldehyde (Biosharp, USA) at room temperature for 10 min and stained with 0.1% crystal violet (Sigma-Aldrich, USA) at room temperature for 15 min. Images were captured by an inverted microscope (Olympus, Japan) and the number of migrated cells was measured using ImageJ 1.53a software (n=3).
4
Adipose Tissue Apoptosis Evaluation
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Native adipose tissue and apoptotic adipose tissue were harvested, fixed in 4% neutral paraformaldehyde (Biosharp, USA), dehydrated in gradient ethanol, transparent in xylene, and finally embedded in paraffin and cut into 5–6 µm thick sections. The morphology was detected by H&E staining (Solarbio, China) according to the manufacturer’s protocol. The apoptosis of adipose tissue was evaluated by TdT-mediated dUTP nick-end labeling (TUNEL) assay, which was usually used for apoptosis detection.17 (link) According to the manufacturer’s instruction of the TUNEL Apoptosis Assay Kit (Beyotime, China), sections were incubated with TUNEL reagent at 37°C for 60 min. Then, DAPI (1:1000, Solarbio, C0050) was used to further mark the nuclei at room temperature for 5 min. Images were captured by confocal microscopy (Olympus FV1000, Japan).
5
Cistanche deserticola Polysaccharide and L-dopa Interactions
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Cistanche deserticola polysaccharide (CDP) andl ‐dopa were purchased from Yuanye Biotec (purity ≥ 98%; Shanghai, China). Hydrogen peroxide (H2O2), dimethyl sulphoxide (DMSO), NaOH, Triton X‐100, 4,5‐dimethylthiazol‐2‐yl‐2,5‐diphenyltetrazolium bromide (MTT) and an Annexin V‐FITC Apoptosis Detection Kit were purchased from Sigma‐Aldrich. 4% neutral paraformaldehyde was purchased from Biosharp (Hefei, China); and an Immunofluorescence Staining Kit (Alexa Fluor 488) and 2,7‐dichlorofluorescein‐diacetate (DCFH‐DA) were purchased from Beyotime Biotec (Shanghai, China). A Fontana‐Masson Stain Kit and a Nucleoplasmic Protein Extraction Kit were purchased from Sloarbio (Beijing, China). Human melanocyte growth supplement (HMGS), Dulbecco's modified Eagle medium (DMEM) and medium 254 were purchased from Gibco. Fetal bovine serum (FBS) was purchased from BI (Kibbutz Beit‐Haemek, Israel). Primary antibodies for β‐actin, TYR, TRP2, RAB27A, FSCN1, ERK, p‐ERK, JNK, p‐JNK, p38, p‐p38, NRF2 and HO‐1 were purchased from Cell Signaling Technology, primary antibody for MITF was purchased from St John's Laboratory, primary antibody for p‐MITF was purchased from Affinity Biosciences, primary antibody for GAPDH was purchased from Bioworld, and primary antibody for TRP1 was purchased from EMD Millipore.
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Cistanche deserticola polysaccharide (CDP) and
6
Sitogluside-Mediated Melanogenesis Regulation
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Sitogluside (purity ≥ 98%) was purchased from ChemFaces (#CFN98, 713, ChemFaces). Dimethylsulfoxide (DMSO) was purchased from Sigma-Aldrich. Neutral paraformaldehyde (4%) was purchased from Biosharp (Hefei, China). Kojic acid, the Fontana-Masson Stain Kit, sodium deoxycholate, and L-DOPA were purchased from Solarbio (Beijing, China). Dulbecco's modified Eagle's medium (DMEM) was purchased from Gibco (#C11995500BT, Gibco). Fetal bovine serum (FBS) and Cell Counting Kit-8 (CCK8) were purchased from BI (Kibbutz Beit-Haemek, Israel). PMA (12-O-tetradecanoyl phorbol-13-acetate, the ERK/MAPK activator) was purchased from APExBIO (#N2060, APExBIO). Anisomycin (the p38/MAPK activator) was purchased from MedChemExpress (HY-18982, MedChemExpress). Primary antibodies against ras-related protein Rab-27A (RAB27A) (#69295, CST), extracellular signal-regulated kinase (ERK) (#4695, CST), p-ERK (#4370, CST), c-Jun N-terminal kinase JNK (#9252, CST), p-JNK (#4668, CST), p38 (#8690, CST), p-p38 (#4511, CST), cAMP response element-binding protein CREB (#9107, CST), and p-CREB (#9198, CST) were purchased from Cell Signaling Technology. Primary antibodies against TRP1 (ab235447, Abcam), TRP2 (NBP1-56058, Novusbio), MITF (STJ94134, St. John's Laboratory), TYR (BS1484, Bioworld), and GAPDH (#AP0066, Bioworld) were purchased from Abcam, Novusbio, St. John's Laboratory, and Bioworld, respectively.
7
Cellular Uptake of ApoEVs-AT
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For cellular uptake, 5×104 HFFs or HUVECs were first seeded into a confocal culture dish (NEST, China). Then, 100 μg ApoEVs -AT, suspended in 1 mL α-MEM, were labeled with 1 μg membrane-labeling dye DiO (Life Tech, V22886) at 37°C for 30 min, re-purified with the centrifugation at 16,000 g for 30 min at 4°C. HFFs or HUVECs were cultured with DiO-labeled ApoEVs-AT for 0, 4, and 8 h. Cells were fixed in 4% neutral paraformaldehyde (Biosharp, USA) at room temperature for 10 min and underwent permeabilization with 0.05% Triton X-100 (Sigma-Aldrich, USA) at room temperature for 15 min. The cytoskeleton was stained with phalloidin (1:200, Invitrogen, A34055-300U) at room temperature for 20 min and the nuclei were stained with DAPI (1:1000, Solarbio, C0050) at room temperature for 5 min. Images were captured by confocal microscopy (Olympus FV1000, Japan).
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