832/13 or 832/3 cells were fixed with 4% formaldehyde in PBS for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min. Cells were blocked in 5% goat serum and 2.5% bovine serum albumin (BSA) in PBS for 1 h and then incubated with the appropriate primary antibodies (1:500 anti-insulin; 1:10,000 anti-Myo1b; and 1:500 anti-TGN38), fluorescently labeled secondary antibodies (1:500), and/or rhodamine- or Alexa Fluor 594–conjugated phalloidin (1:500) to identify actin filaments. For staining of sections from rat pancreas, after deparaffinization and rehydration, the sections were subjected to antigen retrieval by being boiled in 10 mM sodium citrate, pH 6.0, for 5 min. Sections were stained as described above. Sections and cells grown on #1.0 coverslips to be viewed with a Leica TCS SP5 Broadband confocal microscope (Leica, Sohms, Germany) were fixed and mounted on glass slides in Vectashield Hardset antifade mounting medium with DAPI (H-1500; Vector Laboratories, Burlingame, CA). Alternatively, cells grown on #1.5 coverslips were fixed and mounted in ProLong Gold mounting reagent (P10144; Molecular Probes, ThermoFisher Scientific) and viewed with a Nikon N-SIM superresolution system (Minato, Tokyo, Japan) using a 40× or 100× objective.
Vectashield hardset antifade mounting medium with dapi h 1500
Manufactured by Vector Laboratories
Sourced in United States
Vectashield HardSet Antifade Mounting Medium with DAPI (H-1500) is a ready-to-use aqueous mounting medium that contains the nuclear counterstain DAPI. It is designed to retard photobleaching and maintain the fluorescence of labeled specimens.
Automatically generated - may contain errors
Lab products found in correlation
Triton x 100, by Thermo Fisher Scientific (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Lsm 780 upright confocal microscope 63x objective, by Zeiss (2 mentions)
Lsm 510, by Zeiss (2 mentions)
Anti gh2ax antibody 05 636, by Merck Group (2 mentions)
Prolong gold mounting reagent, by Thermo Fisher Scientific (1 mentions)
Axio imager z2 microscope, by Zeiss (1 mentions)
A11012, by Thermo Fisher Scientific (1 mentions)
G10362, by Thermo Fisher Scientific (1 mentions)
Plan apochromat 20 0.8 m27 objective lens, by Zeiss (1 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (1 mentions)
Mouse anti α tubulin, by Merck Group (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Dulbecco s phosphate buffered saline dpbs, by Thermo Fisher Scientific (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Trypsin, by Thermo Fisher Scientific (1 mentions)
Metformin, by Merck Group (1 mentions)
Phenformin, by Merck Group (1 mentions)
8 protocols using vectashield hardset antifade mounting medium with dapi h 1500
1
Immunofluorescence Staining of Cells and Tissue Sections
2
Visualization of TRPM8 in Vascular Cells
3
Immunohistochemical Visualization of GFP in Mouse Brain
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After the loose-seal recording sessions, mice were anaesthetized with a mixture of ketamine–xylazine (0.1 mg ketamine and 0.008 mg xylazine per gram body weight) and were transcardially perfused with 4% PFA in 1X Dulbecco’s PBS (DPBS). The brains were extracted and post-fixed overnight in the perfusing solution. The brains were sectioned at 50-μm thickness, blocked with 2% BSA + 0.4 Triton X-100 (in PBS) for 1 h at room temperature, incubated with primary antibody (Rb-anti-GFP, 1:500; G10362, Invitrogen) for 2 days at 4 °C, and secondary antibody (AlexaFluor 594 conjugated goat anti-Rb, 1:500; A-11012, Invitrogen) overnight at 4 °C. The sections were mounted on microscope slides in Vectashield hard-set antifade mounting medium with DAPI (H-1500, Vector). Samples were imaged using a TissueFAXS 200 slide scanner (TissueGnostics) comprising an X-Light V2 spinning disk confocal imaging system (CrestOptics) built on an Axio Imager.Z2 microscope (Carl Zeiss Microscopy) equipped with a Plan-Apochromat ×20/0.8 M27 objective lens.
4
Visualizing Actin and Tubulin in VSMCs
5
Optimizing Cell Culture Reagents and Tools
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Methanol and ethanol were from VWR International Ltd. (Poole, UK). Metformin (D150959), phenformin (p7045), and other general chemicals were purchased from Sigma-Aldrich (Poole, UK). ECL Western blotting analysis system (RNP2109) was from GE Healthcare (Little Chalfont Bucks). Dulbecco’s Modified Eagle Medium (DMEM) (41965–039, 31885–023), Dulbecco’s phosphate-buffered saline (DPBS) (14190), trypsin (25300–054), penicillin-streptomycin (15140122), lipofectamine 2000, Superscript VILO Master Mix (11755–050), Taqman Universal PCR Master Mix (4324020) were from Thermo-Fisher Scientific (Perth, Scotland). HiPerFect transfection reagent (301704) was from Qiagen (Manchester, UK). Microscopic glass slides (631–0906) were from VWR International (Lutterworth, UK). Vectashield hard-set anti-fade mounting medium with DAPI (H-1500) was from Vector Laboratories Inc. (Peterborough, UK). Details of antibodies (S1 Table ), plasmids (S2 Table , oligos (S3 Table ) and viruses (S2 Table ) are listed in S1 –S3 Tables.
6
Auditory Brainstem Transduction Analysis
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To analyze transduction in the auditory brainstem, brains were dissected out from the injected animals at age postnatal day 9 (P9). Meningeal layers were carefully removed from brain samples, followed by a brief wash in 0.1 M PBS and stored in 4% PFA (in 1X PBS) for 16–24 h. Brain samples were washed 3 times for 10 min with 0.1 M PBS. Coronal auditory brainstem slices of 50-μm thickness containing CN and MNTB from the fixed brain samples were prepared using a Leica VT 1200 vibratome. Free-floating slices were incubated with DAPI (1 μg/mL) for ∼5 min and briefly washed with 0.1 M PBS. Sections then were mounted with Vectashield hardset antifade mounting medium with DAPI (H-1500, Vector laboratories, Burlingame, CA, USA). To analyze transduction in the auditory brainstem. Confocal images were acquired with a Zeiss LSM 880. Auditory brainstem slices were imaged using z stack and tilescan mode (bounding grid) with Plan Neofluar 10X/0.3 and 20X/0.8 and online stitched with overlap 10%–25%. Emission signal intensity for each channel was adjusted to below the saturation level. Images were further processed in Fiji (ImageJ 1.53c) and Adobe Photoshop.
7
Quantification of DNA Damage Response
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Cells were seeded in 4-well chamber slides (Nunc® Lab-Tek® II Chamber Slide™, C6807, Millipore Sigma, St Louis, MO, USA) to 50-60% confluence and allowed to adhere to the slide for 24 h. Cells were then treated with sham or 2 Gy radiation as described in the Irradiation of cell lines and mice section and incubated at 37 °C for 30 min or 24 h prior to washing with PBS and fixation with 3% PFA. Cells were then permeabilized with 0.1% Triton-X and washed with PBS before blocking with blocking buffer (10% FBS, 0.5% BSA in PBS) for 1 h at room temperature. Slides were incubated at 4 °C overnight with 1:300 anti-gH2AX antibody (05-636, Millipore Sigma, St Louis, MO, USA). Slides were washed in three changes of PBS and incubated with 1:500 anti-mouse-IgG AlexaFluor 488 conjugated secondary antibody for 3 h at room temperature. Slides were mounted with VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI
(H-1500, Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained on the Zeiss LSM510. Images were then analyzed using ImageJ Software to quantitate foci per nucleus for a random selection of six 100X fields of view, corresponding to 30–50 cells per condition.
(H-1500, Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained on the Zeiss LSM510. Images were then analyzed using ImageJ Software to quantitate foci per nucleus for a random selection of six 100X fields of view, corresponding to 30–50 cells per condition.
8
Gamma-H2AX Foci Quantification
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Cells were seeded in 4-well chamber slides (Nunc® Lab-Tek® II Chamber Slide™, C6807, Millipore Sigma, St Louis, MO, USA) to 50-60% con uence and allowed to adhere to the slide for 24 hours. Cells were then treated with sham or 2Gy radiation as described in the "Irradiation of cell lines and mice" section and incubated at 37˚C for 30 minutes or 24 hours prior to washing with PBS and xation with 3% PFA. Cells were then permeabilized with 0.1% Triton-X and washed with PBS before blocking with blocking buffer (10% FBS, 0.5% BSA in PBS) for one hour at room temperature. Slides were incubated at 4˚C overnight with 1:300 anti-gH2AX antibody (05-636, Millipore Sigma, St Louis, MO, USA). Slides were washed in three changes of PBS and incubated with 1:500 anti-mouse-IgG AlexaFluor 488 conjugated secondary antibody for 3 hours at room temperature. Slides were mounted with VECTASHIELD® HardSet™ Antifade Mounting Medium with DAPI (H-1500, Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained on the Zeiss LSM510. Images were then analyzed using ImageJ Software to quantitate foci per nucleus for a random selection of six 100X elds of view, corresponding to 30-50 cells per condition.
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