Tumors were cut in half and embedded in optimal cutting temperature compound (OCT) and frozen on dry ice before storing at −80 ˚C. Frozen tissues were the sectioned (5 microns thick) using a cryostat. Tissue slides were fixed in cold methanol in −20 ˚C for 10 min, washed with PBS +0.01% Triton X-100, then pure PBS, blocked with 5% goat serum at room temperature for 1 hour, and stained with purified primary antibody at 4˚C overnight. Tissues were then washed with PBS three times, stained with secondary antibodies at room temperature for one hour, counterstained with Hoechst Dye and mounted in Prolong mounting media (Thermo Fisher Scientific). The following antibodies were used for detecting mouse antigens by immunofluorescent staining: CD4 (eBioscience, clone RM4–5), CD8 (eBioscience, clone 53–6.7), K14 (eBioscience, polyclonal Cat#PA5–16722), K17 (provided by Pierre A Coulombe [57]), Goat anti-rabbit AlexaFluor647 (Molecular Probes), Goat anti-rat AlexaFluor 488 (Molecular Probes).
Prolong mounting media
Manufactured by Thermo Fisher Scientific
Sourced in United States
ProLong mounting media is a high-performance, water-soluble, and non-fluorescent reagent designed for the preservation and mounting of fluorescently-labeled biological samples. It is commonly used to enhance and protect fluorescent signals in microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit alexafluor647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rat, by Thermo Fisher Scientific (1 mentions)
Trehalose, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Axioscope light microscope, by Zeiss (1 mentions)
Deltavision rt, by Cytiva (1 mentions)
8 well microscopy slides, by Ibidi (1 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (1 mentions)
Ox 42, by Bio-Rad (1 mentions)
Neuronal nuclei (neun), by Merck Group (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 or 568, by Thermo Fisher Scientific (1 mentions)
Rabbit anti s 100 protein, by Agilent Technologies (1 mentions)
Hcx pl apo 40, by Leica (1 mentions)
Dm6000b microscope, by Leica (1 mentions)
Phalloidin 488, by Thermo Fisher Scientific (1 mentions)
Axiovert fluorescence microscope, by Zeiss (1 mentions)
Cd144, by Thermo Fisher Scientific (1 mentions)
Jam c, by R&D Systems (1 mentions)
9 protocols using prolong mounting media
1
Cryosectioning and Immunofluorescent Staining
2
Multiplex Immunohistochemistry Staining Protocol
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Primary Abs used for multiplexing (Table 1 Table 2
3
Quantifying Mast Cell Dynamics in Muscle
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Mast cell counts and degranulation was assessed based on previously published methods37 (link). Briefly, longitudinal sections (20um) of tibialis anterior muscle were made from fresh frozen tissue. Sections were washed and hydrated 2 times in distilled water for 10 minutes and embedded in 1% toluidine blue solution (Fisher Scientific, T16125) for 10–20 minutes. Slides were washed in distilled water and dehydrated in 70% ethanol, 95% ethanol, and in 100% ethanol. Slides were cleared in xylene and were mounted in ProLong mounting media (Invitrogen). Images were acquired using a Zeiss Axioscope light microscope. The number of toluidine blue-positive metachromatic mast cells were counted in six individual entire muscle section for each animal. The number of degranulating mast cells was also counted in each of these sections for each animal and were identified by extensive metachromatic granules being released by an isolated cell as described previously37 (link).
4
Quantifying Nix-LC3B Colocalization in HeLa Cells
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HeLa cells were seeded, grown on coverslips or 8-well microscopy slides (ibidi) and transfected with either wild type or mutant Nix or Bnip3 together with wild type or mutant LC3B constructs using JetPRIME transfection reagent according to manufacturer’s instructions. 24 hrs post transfection cells were fixed in 2% or 4% paraformaldehyde and permeabilized with 0,1% Triton X-100 solution. Cells were blocked in 5% BSA/PBS/0.1% Triton X-100 for 1 hr at room temperature or 4 °C overnight. Primary and secondary antibodies were diluted in blocking solution and washed in 0,1% Triton X-100/PBS. Coverslips were mounted in Prolong mounting media (Invitrogen). Cells in 8-well microscopy slides were placed in PBS following staining. Cells were imaged using a Zeiss AxioVision or DeltaVision RT microscope system (Applied Precision). Quantification of Nix-LC3B colocalization was performed as follows: for each Nix construct, LC3B positive dots (signals) were numbered in 100 cells. Only clear and well-defined LC3B signals are taken into consideration, while weak and oversized signals were excluded from the analysis.
5
Immunohistochemical Profiling of Neuronal and Glial Markers
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The serial sections underwent immunohistochemical processing for the identification of neuronal and glial cell markers. Following blocking with normal serum, the following primary antibodies were applied: mouse anti-neuronal-nuclei antibody (NeuN; 1:200, Chemicon), mouse anti-microtubule-associated protein-2 (MAP2; 1:200, Chemicon), rabbit anti-synaptophysin (SYN; 1:500, Dako), rabbit anti-glial fibrillary acidic protein (GFAP; 1:1000, Dako), monoclonal antibodies reacting with C3bi complement receptors (OX42; 1:200, Serotec) and a cocktail of monoclonal antibodies reacting with 68 kDa, 160 kDa and 200 kDa neurofilament proteins (NF; 1:200; Zymed Laboratories). The primary antibodies were applied at room temperature for 2 hours. Following rinsing in PBS, secondary goat anti-mouse and goat anti-rabbit antibodies Alexa Fluor® 488 and Alexa Fluor® 568 (1:300; Molecular Probes, Invitrogen) were applied for 1 h in darkness and at room temperature. All the slides were coverslipped with ProLong mounting media containing DAPI (Invitrogen). The staining specificity was tested by the omission of primary antibodies.
6
Immunophenotyping of Cultured Cells
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Cultured cells were fixed in 4% (w/v) paraformaldehyde for 20 min at room temperature. After blocking with 5% normal horse and goat serum in 0.1% BSA, 0.1% sodium azide, and 0.1% Triton for 15 min, the primary antibody was applied for 2 h at room temperature. The following primary antibodies were used: mouse anti-CD34 (1:200; Chemicon), mouse anti-CD45 (1:200; Chemicon), mouse anti-CD63 (1:200; Chemicon), mouse anti-CD73 (1:200; Chemicon), mouse anti-CD105 (1:200; Chemicon), mouse anti-CD146 (1:200; Chemicon), mouse anti-Thy1.1 (CD90; (1:1000; Chemicon), and rabbit anti-S-100 protein (1:1000; Dako). Cells were then washed with PBS and incubated with normal serum followed by fluorescently conjugated secondary antibodies (Alexa Fluor 488 or 568, 1:300 dilution; Molecular Probes, Invitrogen) for 1 h in the dark. Cells were mounted with ProLong mounting media containing 4′-6-diamido-2-phenylindole (DAPI; Invitrogen Life Technologies).
7
DNA Fiber Analysis of Replication Dynamics
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Cells were pulse-labelled sequentially with 50 μM CldU (20 min) and 250 μM IdU (20 min), harvested and resuspended in PBS (0.5 × 106 cell/ml). 2 μl drops of cell suspension were placed on microscope slides and lysed with 0.5% SDS, 0.2 M Tris pH 7.4, 50 μM EDTA in 10 μl for 6 min at RT. Slides were tilted 15°C to spread DNA fibers, air-dried, fixed in –20°C methanol:acetic acid (3:1) for 2 min and stored at 4°C overnight. Slides were then incubated in 2.5 M HCl (30 min/RT) to denature DNA and washed (3×) in PBS. Blocking solution (1% bovine serum albumin in PBS, 0.1% Triton X-100) was added for 1 h at RT. Slides were incubated with anti-CldU, IdU and ssDNA primary antibodies for 1 h at RT, washed and incubated with the corresponding secondary antibodies for 30 min. Prolong mounting media (Invitrogen) was used. Images were acquired in a DM6000 B Leica microscope with an HCX PL APO 40×, 0.75 NA objective. Fork rate values were derived from the length of IdU tracks, measured using ImageJ software, and a conversion factor of 1 μm = 2.59 kb (36 (link)). >300 tracks were measured per condition. The percentage of origins activated during the first pulse (green-red-green tracks) was quantified relative to all replicative structures containing red signal. >500 total structures were scored in each case. Three biological replicates of each experiment were performed.
8
Immunofluorescence Analysis of Endothelial Junctions
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Treated HDMEC monolayers grown on gelatin-coated glass were washed briefly and fixed in 95% ethanol for 30 min at 4°C. For two-color immunofluorescence imaging, monolayers were incubated overnight in anti–caludin-5 antibody (Invitrogen), ZO-1 (ThermoFisher), CD144 (eBiosciences), CD31 (Life Technologies), JAM-A (R&D Biosystems), and JAM-C (R&D Biosystems) and diluted in Tris-buffered solution/0.2% Triton-X-100/5% normal donkey serum. Donkey Alexa488 and Alexa594 Donkey secondary antibodies along with phalloidin-488 (Life Technologies) were used to detect primary antibody and β-actin. Glass coverslips were mounted for immunofluorescence analysis in ProLong mounting media (Invitrogen). Randomly selected (minimum of five images per experimental condition) fluorescence photomicrographs were collected by using a Zeiss Axiovert fluorescence microscope and Plan-APOCHROMAT, 63× oil objective (Zeiss) with an ORCA-ER digital camera (Hamamatsu Photonics).
9
Oxidative Stress Quantification
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Samples were sectioned in a cryostat (8 μm) and incubated with 150 μM of MnTBAP (Santa Cruz Biotechnology) for 1 h at RT. The samples were then washed with PBS and incubated with 1 μM of dihydroethidium (DHE, Sigma-Aldrich) for 30 min at 37 °C. Sections were washed again and mounted with ProLong mounting media containing DAPI (Invitrogen). Photographs were taken with a fluorescence microscope (Axioimager.D1 Zeiss) and analyzed by ImageJ software.
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