For additional immunofluorescent staining, either CD146+ periosteal pericytes in culture or cryosections (12–16 μm) of human periosteum, adipose, and intramuscular implants were used. All specimens were blocked with 5% serum in PBS for 1 h at 25 °C. Antigen retrieval was by trypsin enzymatic antigen retrieval solution (Cat# ab970; Abcam) for 5 min. Specimens were incubated with the following primary antibodies: anti-CD146 (Abcam, 1:50), anti-CD31 (Cell Signaling Technology, 1:320 or Abcam, 1:100), anti-Gli1 (Abcam, 1:100), anti-PDGFRα (Abcam, 1:200), anti-PDGFRβ (Abcam, 1:100), anti-OCN (Abcam, 1:200), and anti-CXCR4 (Abcam, 1:150). The Dylight 594 goat anti-rabbit IgG (H + L) polyclonal (Vector, 1:200), Goat anti-armenian hamster IgG (H+L) Alexa Fluor 647 (Abcam, 1:200) or Goat anti-mouse IgG (H + L) Alexa Fluor 488 or 647 (Abcam, 1:200) were used as secondary antibodies. Sections were counterstained with DAPI mounting medium (Cat# H-1500, Vector laboratories). All histological sections were examined with a Zeiss 780 confocal microscope (Zeiss, Thornwood, NY, USA) or microscopy of Leica DM6 B (Leica Microsystems Inc).
Trypsin enzymatic antigen retrieval solution
Manufactured by Abcam
Sourced in United Kingdom
Trypsin enzymatic antigen retrieval solution is a laboratory reagent used to expose target antigens in fixed tissue samples prior to immunohistochemical staining. It functions by enzymatically digesting proteins, facilitating the accessibility of the target epitopes for antibody binding.
Automatically generated - may contain errors
Lab products found in correlation
780 confocal microscope, by Zeiss (1 mentions)
Anti pdgfrβ, by Abcam (1 mentions)
Dm6 b, by Leica (1 mentions)
Anti ocn, by Abcam (1 mentions)
Dapi mounting medium, by Vector Laboratories (1 mentions)
Anti pdgfrα, by Abcam (1 mentions)
Anti gli1, by Abcam (1 mentions)
Anti cd146, by Abcam (1 mentions)
Anti cd31, by Cell Signaling Technology (1 mentions)
Anti cxcr4, by Abcam (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions)
Il 10, by Thermo Fisher Scientific (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Proteinase k, by Merck Group (1 mentions)
Monensin, by Merck Group (1 mentions)
Ifn γ, by Thermo Fisher Scientific (1 mentions)
Mouse monoclonal ab against socs3, by Abcam (1 mentions)
Mouse monoclonal ab against β actin, by Merck Group (1 mentions)
4 protocols using trypsin enzymatic antigen retrieval solution
1
Immunofluorescent Analysis of Periosteal Cells
2
Investigating SOCS Signaling in Cell Cultures
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RPMI 1640 and F12-K were purchased from Gibco-Invitrogen. PGE2 from Cayman Chemical was dissolved in DMSO and stored under N2 at −80°C. Murine and rat cytokines (IL-6, IFNγ, and IL-10) were purchased from PeproTech. Mouse monoclonal Ab against SOCS3 and rabbit polyclonal Ab against SOCS1 were from Abcam and Cell Signaling Technology, respectively. Mouse monoclonal Ab against β-actin was from Sigma-Aldrich. FITC-conjugated rabbit polyclonal Abs against SOCS3 and SOCS1 were from Biorbyt. The fluorescent lipid 1-oleoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (18:1-06:0 NBD PC) was purchased from Avanti Polar Lipids, Inc. Rabbit polyclonal Abs against phospho- and total STAT1 and STAT3 were from Cell Signaling Technology. LPS, monensin, hematoxylin, and proteinase K were from Sigma-Aldrich. Trypsin enzymatic antigen retrieval solution was from Abcam. Compounds requiring reconstitution were dissolved in PBS, EtOH, or DMSO. Required dilutions of all compounds were prepared immediately before use, and equivalent quantities of vehicle were added to the appropriate controls. DMSO or EtOH at the concentrations used had no direct effect on SOCS3 secretion.
3
Quantifying Sclerostin Expression in Bone
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To assess sclerostin expression in tibiae, tibiae sections were deparaffinized in xylene and rehydrated through ethanol series. Sections were incubated with Trypsin Enzymatic Antigen Retrieval Solution (Abcam, Cambridge, UK) for 15 min at 37 °C followed by immersion in 3% H2O2 in methanol for 20 min to block endogenous peroxidase. After blocking in normal horse serum (Vector Laboratories, Burlingame, CA, USA) for 20 min, the sections were incubated overnight at 4 °C with goat anti-sclerostin antibody (1:50 dilution, R&D Systems, Minneapolis, MN, USA). After incubation with anti-goat secondary antibody (Vector Laboratories) for 20 min, sections were developed with 3,3-diaminobenzidine tetrahydrochloride substrate chromogen system (DAKO, Botany, Australia) and counterstained with methyl green. Slides were scanned as described above. The number of sclerostin-positive osteocytes in the ROI was divided by the number of total osteocytes in the ROI. The ROI was the same as the ROI for osteoclast counting.
4
Evaluation of CELF1 Expression in Breast Tissue
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Breast cancer tissue microarray containing tissue samples from 150 breast cancer patients (HBre-Duc150Sur-01), breast tissue array containing 90 normal adjacent breast tissue samples (HBre-Duc090Sur-01), and high-density multiple organ tumour tissue array with normal tissue as control (MC5003b) were obtained from US Biomax (Rockville, MD, USA). The TMAs were stained for CELF1 expression using routine methodology using the trypsin enzymatic antigen retrieval solution (Abcam) and the anti-CUGBP1 antibody [3B1] (ab9549; Abcam) at a 1:500 dilution. The stained slides were scored by a pathologist (D.G.R.) blinded to the identity of the tissue cores as per cent of CELF1-positive cells with a range of 0 to 100). Tissue cores representing stromal (non-ductal) tissue were excluded from analysis. For the tumour-normal pairs the scores in both samples and their changes (tumour-normal) were summarized.
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