One‐time centrifuged adipose tissue and tSVF (controls) as well as acellular matrix of nondiabetic (NAM) and diabetic patients (DAM; n = 3) were formalin fixed and embedded in paraffin. Four‐micrometre thickness sections were cut, deparaffinized, and then incubated overnight with 0.1 M of Tris/HCL buffer (pH 9.0) at 80°C and stained with antibody against Perilipin A (1:200, Abcam) to visualize adipocytes as previously described (van Dongen, Stevens, Parvizi, van der Lei, & Harmsen, 2016 ). Samples were visualized under a light microscope (Leica Microsystems, DM IL). A Masson's trichrome staining and haematoxylin and eosin (H&E) staining were performed on deparaffinized 4 μm of slides. Then samples were mounted and visualized under a light microscope (Leica Microsystems, DM IL).
Perilipin a
Manufactured by Abcam
Sourced in United Kingdom
Perilipin A is a protein that plays a central role in the regulation of intracellular lipid storage and metabolism. It coats the surface of lipid droplets and controls the access of lipases to the droplet core, thereby modulating lipolysis.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Insulin, by Agilent Technologies (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Rhodopsin, by Abcam (1 mentions)
K79bb10, by Merck Group (1 mentions)
Peripherin, by Abcam (1 mentions)
Phosducin, by Santa Cruz Biotechnology (1 mentions)
Gtu 88, by Merck Group (1 mentions)
Gamma tubulin, by Merck Group (1 mentions)
Hsp90, by Santa Cruz Biotechnology (1 mentions)
Glucagon, by Merck Group (1 mentions)
Gm130, by BD (1 mentions)
Imager z2, by Zeiss (1 mentions)
Tdtomato, by Abcam (1 mentions)
Calcein pi staining kit, by Beyotime (1 mentions)
Alpha smooth muscle actin α sma, by Abcam (1 mentions)
F4 80, by Abcam (1 mentions)
Alexa fluor 555, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Desmin, by Cell Signaling Technology (1 mentions)
5 protocols using perilipin a
1
Histological Analysis of Adipose Tissue and Extracellular Matrix
2
Immunofluorescence Labeling of Diverse Cellular Markers
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Aquaporin-2, rabbit polyclonal (Sigma); Gamma tubulin, mouse monoclonal clone GTU-88 (Sigma); Arl13b, mouse monoclonal clone N295B/66 (NeuroMab, University of California Davis); T1α, hamster monoclonal clone 8.1.1 (Developmental Studies Hybridoma Bank, University of Iowa); Perilipin-A, goat polyclonal (Abcam, Cambridge, MA); Insulin, guinea pig polyclonal (DAKO); Glucagon, mouse monoclonal clone K79bB10 (Sigma); Helix pomatia agglutinin, Alexa488 conjugated (Thermo Fisher Scientific); Lotus Tetragonolobus Agglutinin, fluorescein labeled (Vector Labs, Burlingame, CA); Arf4, rabbit polyclonal antibody against residues 98–114 of mouse Arf4; Rhodopsin, monoclonal clone 1D4 (Abcam); Peripherin, rabbit polyclonal antibody (Gabriel Travis, University of California Los Angeles); Phosducin, mouse monoclonal clone G-7 (Santa Cruz, Dallas, TX); GM130, mouse monoclonal clone 35 (BD Biosciences, San Jose, CA); B-actin, mouse monoclonal clone C4 (Santa Cruz); Hsp90, rabbit polyclonal (Santa Cruz).
3
Histological Characterization of Skin Lesions
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Skin lesions were fixed in paraformaldehyde for at least 48 h and embedded in paraffin. Sections (5 µm) were dewaxed using graded xylene and ethanol. Hematoxylin and eosin (H&E) as well as Masson’s trichrome (MT) staining was conducted according to the manufacturer’s instructions. CD31 (1:500; Abcam, Cambridge, UK), alpha smooth muscle actin (α-SMA, 1:200; Abcam) and perilipin A (1:800; Abcam) were used for immunohistochemical assays. For immunofluorescence assay, sections were incubated with following antibodies: F4/80 (1:100; Abcam), GFP (1:250; Abcam), Tdtomato (1:250; Abcam), Sox9 (1:200; Abcam) and then goat anti-chicken secondary antibody, Alexa Fluor 488 (1:500; Thermo Fisher Scientific, MA, USA) and goat anti-rabbit secondary antibody, Alexa Fluor 555 (1:500; Thermo Fisher Scientific). The cell viability was tested according to Calcein/PI staining kit (Beyotime Biotechnology, Shanghai, China). Images were obtained using an Olympus BX51 microscope (Olympus Corporation, Tokyo, Japan) or fluorescence microscope (Imager Z2, Carl Zeiss) and analyzed using the ImageJ software. Number of hair follicles and sebaceous glands was count manually on the samples with H&E staining.
4
Oxidative Stress and Autophagy Modulation
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Glutathione (GSH), N-acetyl cysteine (NAC), hydrogen peroxide (H2O2), sodium pyruvate (Na Py), catalase (Cat), superoxide dismutase (SOD), chloroquine (CQ), 3-methyl adenine (3-MA), Bafilomycin A1, dimethyl sulfoxide (DMSO), Mito Peroxy Yellow 1 (MitoPY1), anti-rabbit IgG, and anti-mouse IgG were purchased from Sigma-Aldrich (St Louis, MO, USA). Dulbecco's modified essential medium (DMEM), Opti MEM medium, phosphate buffered saline (PBS), trypsin-EDTA, and fetal bovine serum (FBS) were bought from GIBCO BRL (Grand Island, NY, USA). Oxidation sensitive 2′,7′-dichlorodihydrofluorescin diacetate (DCFH-DA) was purchased from Molecular Probes (Eugene, OR, USA). Primary antibodies against α-SMA, Desmin, GFAP, LC3-I/II, ULK1, p-ULK1, mTOR, p-mTOR, Atg5, Atg7, Atg9, Atg14, P62, PI3 Kinase Class III (PI3KCIII), and β-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against α1(I) procollagen was purchased from Epitomics (San Francisco, CA). Primary antibodies against Perilipin A, Rab25, and Fibronectin were purchased from Abcam Technology (Abcam, Cambridge, UK). Atg5 siRNA, Rab25 siRNA, negative control siRNA, Atg5 plasmid constructs and negative control vectors were purchased from Hanbio (Shanghai, China). MegaTran 1.0 transfection reagent was from OriGene (Rockville, MD, USA).
5
Histological Characterization of tSVF
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Samples of tSVF were formalin-fixed and embedded in paraffin. Then, 4 μm slides were cut, deparaffinized, and stained following the protocol previously published by van Dongen et al. 19 Samples were stained for α-smooth muscle actin (α-SMA, 1:200; Abcam, Cambridge, UK) to stain smooth muscle cells, von Willebrand factor (vWF, 1:200; DAKO, Glostrup, Denmark) to stain endothelial cells, and perilipin A (1:200, Abcam) to stain adipocytes. As secondary antibodies, polyclonal rabbit anti-mouse for α-SMA, polyclonal swine anti-rabbit for vWF, and polyclonal goat anti-rabbit for perilipin A were used in 1:100 (DAKO).
A third antibody was used for the α-SMA staining (polyclonal, swine anti-rabbit, 1:100; DAKO). Masson's trichrome staining was used to stain extracellular matrix deposition.
A third antibody was used for the α-SMA staining (polyclonal, swine anti-rabbit, 1:100; DAKO). Masson's trichrome staining was used to stain extracellular matrix deposition.
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