Skin sections underwent deparaffinization and were permeabilized with 0.5% Triton X-100 in PBS for 5 min. They were next incubated with blocking buffer (5% normal donkey serum, 0.2% Triton-X in PBS) prior to primary antibody incubation at 4 °C overnight with the following antibodies diluted in blocking buffer: anti-TGF-β(1:400, MAB240-SP, R&D system, MN, US), anti-Collagen I (1:200, ab24821, Abcam, Cambridge, UK), and anti-Collagen III (1:400, ab6310, Abcam). After three 30-min washes with PBS + 0.05% Triton-X, samples were stained for 1 h at room temperature with fluorescent secondary antibodies (ThermoFisher Scientific, MA, USA) followed by two washes with PBS. The slides were mounted with Antifade Mounting Medium with DAPI (h-1200, Vector laboratories, CA, USA) and viewed under a spinning-disc confocal microscope (Carl Zeiss).
Antifade mounting medium with dapi h 1200
Manufactured by Vector Laboratories
Sourced in United States
Antifade Mounting Medium with DAPI (h-1200) is a product from Vector Laboratories. It is a mounting medium that contains an antifade agent and the fluorescent dye DAPI, which binds to DNA.
Automatically generated - may contain errors
Lab products found in correlation
Spinning disc confocal microscope, by Zeiss (1 mentions)
Ab6310, by Abcam (1 mentions)
Fluorescent secondary antibody, by Thermo Fisher Scientific (1 mentions)
Chloroquine cq, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
2 7 dichlorodihydrofluorescein diacetate h2dcfda, by Thermo Fisher Scientific (1 mentions)
N acetyl cysteine (nac), by Santa Cruz Biotechnology (1 mentions)
Z vad fmk, by Abcam (1 mentions)
Sorafenib tosylate, by Santa Cruz Biotechnology (1 mentions)
Bx43 microscope, by Olympus (1 mentions)
Cellsens dimension software, by Olympus (1 mentions)
Ht25a, by Merck Group (1 mentions)
3 protocols using antifade mounting medium with dapi h 1200
1
Immunofluorescent Staining of Skin Sections
2
Evaluation of Sorafenib Cytotoxicity Mechanisms
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Reagents used in the present study were as follows: Sorafenib Tosylate (475207-59-1; Santa Cruz Biotechnology, Santa Cruz, California, USA); Caspase family inhibitor Z-VAD-FMK (1010-100; BioVision, CA USA); Cycloheximide (CHX), a protein synthesis inhibitor (C1988; Sigma-Aldrich, St. Louis, Missouri, USA); antioxidant N-acetyl-cysteine (NAC) (Santa Cruz Biotechnology, Santa Cruz, California, USA); Human serum albumin (ALB) (A1653; Sigma-Aldrich, St. Louis, Missouri, USA), an autophagy inhibitor Chloroquine (CQ) (H0915; Sigma-Aldrich, St. Louis, Missouri, USA), 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) (D399; Invitrogen, San Diego, CA, USA), 3-Ethoxy-5,6-dibromosalicylaldehyde (EDBS) (SML0149; Sigma-Aldrich, St. Louis, Missouri, USA), Antifade Mounting Medium with DAPI (H-1200; Vector Laboratories, San Diego, CA), Propidium Iodide (PI) (P4170; Sigma-Aldrich, St. Louis, Missouri, USA) and dimethyl sulfoxide (DMSO) (D2650; Sigma-Aldrich St. Louis, Missouri, USA).
3
Histological and Immunofluorescence Analysis of Aortic Tissue
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For aortic histology, aortas were excised as above and fixed in formalin (10%) overnight before embedding in paraffin. Sections (5 µm) were stained with hematoxylin and eosin, Masson’s trichrome stain for collagen deposition (Newcomers Supply; 9179B), or Verhoeff–Van Gieson stain for elastin (Sigma; HT25A). Images were captured using Olympus BX43 microscope and Olympus cell Sens Dimension software.
For immunofluorescence, formalin-fixed, paraffin-embedded tissue slides obtained from patients with AAA and aortic healthy control were heated for 30 min at 60°C, deparaffinized, and rehydrated. Slides were placed in pH 9 antigen retrieval buffer and heated at 125°C for 3 s in a pressure-cooker water bath. After cooling, slides were blocked using 10% donkey serum (30 min). Overnight coincubation (4°C) was then performed using anti-human JMJD3 (Abcam; catalog no. ab3811), anti-human CD11c (Abcam; ab216028), and isotype control at a concentration of 1 µg/ml. Slides where then washed and treated with relative fluorescence-conjugated secondary antibodies (30 min). Slides were prepared in mounting medium with DAPI (Vector; VectaShield, Antifade Mounting Medium with DAPI, H-1200). Images were acquired using a Zeiss Axioskop 2 microscopy. Images presented are representative of at least three biological replicates.
For immunofluorescence, formalin-fixed, paraffin-embedded tissue slides obtained from patients with AAA and aortic healthy control were heated for 30 min at 60°C, deparaffinized, and rehydrated. Slides were placed in pH 9 antigen retrieval buffer and heated at 125°C for 3 s in a pressure-cooker water bath. After cooling, slides were blocked using 10% donkey serum (30 min). Overnight coincubation (4°C) was then performed using anti-human JMJD3 (Abcam; catalog no. ab3811), anti-human CD11c (Abcam; ab216028), and isotype control at a concentration of 1 µg/ml. Slides where then washed and treated with relative fluorescence-conjugated secondary antibodies (30 min). Slides were prepared in mounting medium with DAPI (Vector; VectaShield, Antifade Mounting Medium with DAPI, H-1200). Images were acquired using a Zeiss Axioskop 2 microscopy. Images presented are representative of at least three biological replicates.
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