Cells grown on Nunc chamber slides were fixed with 4% paraformaldehyde and permeabilized in PBS containing 0.5% Triton X-100 and blocked in Block-Aid (Life Technologies) for 1 h at room temperature. Cells were then incubated with MF20 monoclonal antibody (DSHB) against MHC (1:40 dilution;) overnight at 4 degrees. Secondary antibody Alexa Fluor 488-conjugated secondary antibody (1:200 dilution; Life Technologies) for 1 h at room temperature. Cells were mounted with ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole; Life Technologies).
Prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi
Manufactured by Thermo Fisher Scientific
Sourced in United States, Japan, Germany, Italy
ProLong Gold antifade reagent with 4′,6-diamidino-2-phenylindole (DAPI) is a mounting medium used to preserve fluorescence in microscopy samples. It contains an antifade agent and the DNA-binding dye DAPI, which labels nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (7 mentions)
Triton x 100, by Merck Group (6 mentions)
Bovine serum albumin (bsa), by Merck Group (6 mentions)
Alexa fluor 488 phalloidin, by Thermo Fisher Scientific (6 mentions)
Lsm 710 confocal microscope, by Zeiss (6 mentions)
Lsm 710, by Zeiss (6 mentions)
Lsm800 confocal microscope, by Zeiss (6 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Paraformaldehyde (pfa), by Merck Group (4 mentions)
Goat serum, by Merck Group (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Image pro plus software, by Media Cybernetics (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Lsm 780 confocal microscope, by Zeiss (3 mentions)
Sp8 confocal microscope, by Leica (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Merck Group (2 mentions)
Leupeptin, by Merck Group (2 mentions)
Tween 20, by Thermo Fisher Scientific (2 mentions)
136 protocols using prolong gold antifade reagent with 4 6 diamidino 2 phenylindole dapi
1
Immunofluorescent Staining of Myosin Heavy Chain
2
Multiplex Immunofluorescence Staining of Tissue Sections
3
Visualizing AVPR2 Receptor Expression
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The Flag-tagged wild-type AVPR2 and AVPR2-∆V279 plasmids were generated by subcloning each target gene’s full-length cDNA into the BamHI XhoI sites of a pCMV-3Tag-1A vector (Yao-Hong Biotechnology, New Taipei City, Taiwan). Human embryonic kidney 293T (HEK293T) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin (100 U/mL)/streptomycin (100 μg/mL) (Thermo Fisher Scientific, Waltham, MA, USA). 293T cells (5 × 104) were co-transfected with 2 μg of wild-type or mutant AVPR2 and the CherryPicker Cell Capture plasmids (Takara Bio, Shiga, Japan) using a calcium phosphate transfection procedure [19 (link)]. The Flag-tagged epitope-specific mouse monoclonal antibody (Sigma-Aldrich, St. Louis, MO, USA) was used for immune-staining, followed by phosphate buffered saline (PBS) washing. Then, Flag-tagged wild-type or mutant AVPR2 was visualized using a rhodamine-conjugated secondary antibody (Jackson ImmunoResearch, Philadelphia, PA, USA). ProLong® Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Life Technologies, Carlsbad, CA, USA) was used for nucleus staining. Fluorescence confocal microscopy was performed using a confocal microscope set, CARV II™ Confocal Imager, equipped with an inverted Olympus IX71S8F3 optical microscope.
4
Histological Analysis of Temporomandibular Joint
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The right PFA-fixed hemi-mandible was decalcified with 14% EDTA (pH 7.5) for up to 14 days, depending upon the animal’s age. Samples were then paraffin embedded so that the condyle could be sectioned in the sagittal or coronal plane. Six μm thick sections were obtained and stained with hematoxylin and eosin (H&E) or toluidine blue using standard methods. Sections from equivalent regions of the TMJ were compared between animals.
For experiments assessing the efficiency of EdU labeling in WT mice and of Cre-mediated recombination in AcanCreERt2/+; ROSA26mTmG/+ mice, after the mandible was decalcified in EDTA, it was equilibrated in 15% followed by 30% sucrose before being embedded in O.C.T. compound (Sakura, Torrance, CA). Eight μm thick frozen sections from WT and AcanCreERt2/+; ROSA26mTmG/+ mice were then obtained in the sagittal plane. For EdU containing sections, the Alexa Fluor 647 Click-iT EdU imaging kit (Invitrogen, Carlsbad, CA) was using following the manufacturer’s directions. All frozen sections were stained by ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes, Eugene, OR) and imaged using a Nikon Eclipse 80i (Nikon, Sendai, Japan) fluorescence microscope with a CoolSNAP HQ2 camera (Photometrics, Tucson, AZ).
For experiments assessing the efficiency of EdU labeling in WT mice and of Cre-mediated recombination in AcanCreERt2/+; ROSA26mTmG/+ mice, after the mandible was decalcified in EDTA, it was equilibrated in 15% followed by 30% sucrose before being embedded in O.C.T. compound (Sakura, Torrance, CA). Eight μm thick frozen sections from WT and AcanCreERt2/+; ROSA26mTmG/+ mice were then obtained in the sagittal plane. For EdU containing sections, the Alexa Fluor 647 Click-iT EdU imaging kit (Invitrogen, Carlsbad, CA) was using following the manufacturer’s directions. All frozen sections were stained by ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Molecular Probes, Eugene, OR) and imaged using a Nikon Eclipse 80i (Nikon, Sendai, Japan) fluorescence microscope with a CoolSNAP HQ2 camera (Photometrics, Tucson, AZ).
5
Immunocytochemical Characterization of Single Cells
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Cytospins of SCS were fixed with methanol. The DakoCytomation EnVision Doublestain system was used for chromogenic immunocytochemistry. For fluorescent immunocytochemistry, cytospins were blocked with Image-iT FX, incubated with primary antibodies (anti-CD3 [polyclonal], Dako; anti-CD1a [O10], Abcam), washed, labeled with secondary antibodies (Alexa Fluor 488 and Alexa Fluor 596, Invitrogen), washed and mounted with ProLong Gold antifade reagent with DAPI (4,6 diamidino-2-phenylindole) (Invitrogen Molecular Probes). Isotype-matched antibodies were used as negative controls.
6
Immunofluorescence Assay for MDR1 Protein
7
Lopinavir and Miconazole Assay Protocol
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Lopinavir was synthesized in the Laboratory of Chemical Synthesis, Farmanguinhos, FIOCRUZ, and dissolved in dimethyl sulfoxide (DMSO). Miconazole, heat-inactivated fetal bovine serum (FBS), 7-hydroxy-3H-phenoxazin-3-one-10-oxide sodium salt (Resazurin sodium salt), RPMI-1640 medium, adenine, d -biotin, folic acid, streptomycin, penicillin, hemin, poly-l -lysine, DMSO and lipid standards were purchased from Sigma Aldrich Chemical (St. Louis, MO, USA). ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) and BODYPI were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Silica plates 60 F254 for thin-layer chromatography (TLC) were purchased from Merck (Frankfurt, DS, Germany). All solvents used were of the purest grade available. All other reagents were analytical grade or superior.
8
Immunofluorescence Staining of Myosin Heavy Chain
9
Immunofluorescence Imaging of E. chaffeensis Interactions
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Uninfected or E. chaffeensis-infected cells or THP-1 cells stimulated with TRP120- or /thioredoxin-coated beads and TRP120 in soluble form were cytospun onto glass slides, fixed for 15 min using 3% paraformaldehyde in phosphate-buffered saline (PBS), blocked and permeabilized for 30 min using 0.3% Triton X-100 and 2% bovine serum albumin (BSA) in PBS at room temperature. Cells were then incubated with primary antibodies rabbit anti-TRP120 (1:1,000), dog anti-E. chaffeensis serum (1:100), goat anti-NICD (1:50), mouse anti-ADAM17 (1:50), goat anti-Notch1 (1:50), rabbit anti-Hes1 (1:50), mouse anti-TLR2 (1:50), mouse anti-TLR4 (1:50), and mouse anti-PU.1 (1:50) for 1 h, washed, and incubated with Alexa Fluor 488 IgG (H+L) and Alexa Fluor 568 IgG (H+L) secondary antibodies (1:100 [Molecular Probes]) for 30 min. Slides were mounted with ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen) after washing. HeLa cells transfected with the GFP-TRP120 plasmids or GFP control plasmids were fixed in chamber slides, permeabilized, and stained with anti-ADAM17 using the same protocol. Images were obtained using an Olympus BX61 epifluorescence microscope and analyzed using Slidebook software (version 5.0; Intelligent Imaging Innovations, Denver, CO).
10
Multimodal Immunostaining for Cellular Markers
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For BrdU staining, all sections were washed with PBS three times, denatured (2 N HCl) for 1 h, neutralized with 0.1 M boric acid, pH 8.5 for 20 min and washed with PBS three more times. Sections were then blocked in 10% (v/v) NGS (normal goat serum)/0.5% (v/v) Triton X-100/1X PBS for 1 h before incubation with rat anti-BrdU (1:200; Accurate), with or without cell-specific antisera for 36–48 h at 4°C. The following antisera against cell-specific markers were used: rabbit anti-NG2 (1:400, Millipore), rabbit anti-GFAP (glial fibrillary acidic protein) (1:1000, DAKO), rabbit anti-Iba-1 (1:400, WAKO) rabbit anti-CD11b (1:200, Serotec), mouse anti-S100β (1:500, Sigma) and rabbit anti-DCX (doublecortin) (1:200, Santa Cruz). Sections were washed three times in PBS and incubated with the corresponding Alexa Fluor 488 or 568-conjugated IgG secondary antibodies (all 1:100; Jackson Immunoresearch) for 1 h at room temperature. Sections were rinsed with PBS, mounted on to the slides and coverslipped with ProLong Gold antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) (Invitrogen).
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