Mouse hippocampal HT22 cells were seeded in 8-well chamber slides at 15,000 cells per well. After 24 h, cells were incubated for 6 h in EBSS medium–containing 10 μg/ml of DQ-BSA red dye, supplemented with 30 μM of UA or vehicle and/or 50 μM of bafilomycin. Cells were then fixed with 4% PFA prepared in PBS for 15 min, washed three times with PBS, permeabilized for 10 min in PBS plus 0.1% Triton X-100 and mounted with ProLong Gold plus DAPI (ThermoFisher, #P36931).
Prolong gold plus dapi
Manufactured by Thermo Fisher Scientific
ProLong Gold plus DAPI is a mounting medium formulated for fluorescence microscopy. It contains an antifade reagent and the DNA-binding dye DAPI to stain nuclei.
Automatically generated - may contain errors
Lab products found in correlation
Gfap cy3, by Merck Group (2 mentions)
Nb100 1613, by Abcam (2 mentions)
Goat anti chicken 633, by Thermo Fisher Scientific (2 mentions)
Ab16048, by Abcam (2 mentions)
Vt1000s vibratome, by Leica (1 mentions)
Dako antibody diluent, by Agilent Technologies (1 mentions)
Tcs sp5 x confocal microscope, by Leica (1 mentions)
Cintiq 22hd art tablet, by Wacom (1 mentions)
Dako target retrieval solution, by Agilent Technologies (1 mentions)
Goat anti rabbit alexa 488, by Thermo Fisher Scientific (1 mentions)
Lsm 780 confocal microscope, by Zeiss (1 mentions)
Alexa fluor conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bromodeoxyuridine (brdu), by Thermo Fisher Scientific (1 mentions)
Fluoromount, by Thermo Fisher Scientific (1 mentions)
Histochoice, by Avantor (1 mentions)
Axio imager m2 microscope, by Zeiss (1 mentions)
Af2125, by R&D Systems (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Polyjet, by SignaGen (1 mentions)
Mitotracker red fm, by Thermo Fisher Scientific (1 mentions)
18 protocols using prolong gold plus dapi
1
Visualizing Autophagy in HT22 Cells
2
Immunohistochemical Analysis of Alzheimer's Pathology
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Anesthetized mice were perfused with 1 × PBS and whole brains were sagittally bisected. Left hemispheres were placed in 4% PFA for 48 h, then washed in cold TBS and cut using a Leica VT 1000S Vibratome. Fifty-micrometer coronal sections were obtained and stored in cryogenic buffer (0.1 M potassium acetate, 40% ethylene glycol, and 1% polyvinyl pyrrolidone) at − 20 °C. Free-floating sections showcasing the rostral hippocampus were subject to epitope unmasking in 88% formic acid for 3 min, then transferred to 500-μL citrate buffer (0.01 M, 6.0 pH) and microwaved 3 × 5 s at 100% power. Sections were then washed 3 × in TBS, permeabilized for 1 h in TBS plus 0.2% Triton X-100, blocked for 1 h in 5% normal goat serum in TBS 0.2% Triton X-100 permeabilization buffer for 1 h. Subsequently, sections were incubated in blocking buffer at 4 °C overnight with anti-amyloid beta peptide antibody (MOAB-2) (1:200) or at room temperature overnight with anti-phospho-Tau AT8 antibody (1:50). Sections were washed 3 × with TBS plus 0.1% Tween-20 for 1 h and incubated in TBS, 0.1% Tween-20, 5% NGS, and goat anti-mouse IgG (H + L) secondary antibody, Alexa Fluor™ 488 (1:500). After 3 additional washes with TBS plus 0.1% Tween-20, sections were mounted on glass slides with ProLong Gold plus DAPI (ThermoFisher, #P36931).
3
Quantifying Mitochondrial Morphology in FFPE Liver
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FFPE liver sections were deparaffinized followed by blocking of endogenous peroxidases. Antigen retrieval was carried out by heating samples at 119 C for 6.5 min in citrate buffer solution (Dako Target Retrieval Solution, Agilent Technologies, Santa Clara, CA, United States). After blocking with 1.5% horse serum, slides were incubated overnight in anti-HSP60 (D6F1, Cell Signaling Technology, Danvers, MA, United States) in Dako antibody diluent (S3022, Agilent Technologies). Secondary antibody incubation with goat anti-rabbit Alexa 488 (ThermoFisher) was carried out at room temperature for 1 h. Slides were mounted using Prolong Gold plus DAPI (ThermoFisher). Slides were imaged using a Leica TCS SP5 X confocal microscope. Confocal images were viewed in a blinded fashion using 3Dmod software on a Wacom Cintiq 22HD art tablet (Vancouver, WA, United States). Mitochondrial length and number per total cytoplasmic area were quantified from ≥ 15 images per group (n ≥ 3 biological replicates) via manual tracing of cell boundaries, nuclei, lipid droplets and mitochondria. Total cytoplasmic area was calculated as area within the cell boundary minus the nuclei and lipid droplet areas.
4
Immunohistochemistry of Midbrain Dopaminergic Neurons
5
Immunohistochemistry of Midbrain Dopaminergic Neurons
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SNpc sections (6 μM) were rehydrated as follows: 2 × 7 min, xylene; 4 min each; 100%, 95%, 80%, and 70% ETOH; rinsed in dH20; and washed in Tris-buffered saline (TBS), 10 min. Slides were then microwaved in 10 mM citrate buffer (pH 6.0) for 5 min at 40% power, cooled for 20 min, washed 10 min in TBS, and then blocked with 10% normal goat serum (NGS) in TBS + 0.05% Triton X-100 for 1 hr at room temperature. Sections were incubated at 4°C overnight with primary antibodies in blocking buffer (chicken TH: Novus, NB100-1613, 1:200 dilution; lamin B1: Abcam, ab16048, 1:200 dilution). Slides were washed three times in TBS followed by TBS, 0.025% Tween-20, 5% normal goat serum, and fluorescent conjugated antibodies (goat anti-rabbit-488: Life Technologies, A31566, 1:500 dilution; goat-anti chicken 633: Life Technologies, A21103, 1:500 dilution; and GFAP-cy3: Sigma, C9205, 1:2,000 dilution). Slides were washed three times in TBS and mounted with ProLong Gold plus DAPI (Life Technologies, P36934).
6
Detection of GlyRα1 Autoantibodies in Patient Sera
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Twenty-four hours after transient transfection, patient or healthy control serum (heat inactivated [56°C] to deplete complement) was added to the GlyRα1-transfected cells and incubated for an additional 4 hours at 37°C.1 (link),7 (link) Slides were then washed twice at 4°C. The positive control serum was diluted in 20% normal goat serum and added to the cells (on ice). After 30 minutes, cells were washed in cold PBS, and goat anti-human IgG-FITC secondary antibody (Southern Biotechnology Associates, Inc.) was added and incubated for 30 minutes. Cells were washed and fixed in 4% paraformaldehyde for 15 minutes. After further washing, cells were mounted in Prolong Gold plus DAPI (Molecular Probes) and imaged (Zeiss LSM780 confocal microscope). ImageJ software was used for quantitation.
7
Quantifying Connexin 32 Expression and Cell Proliferation
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Transfected cells were grown overnight after seeding on to glass coverslips, treated as described, and fixed 15-min at room temperature with Histochoice (Amresco). Coverslips were washed with PBS supplemented with 0.05% Tween. Blocking and antibody dilutions were carried out in PBS supplemented with 0.05% Tween and 2% BSA. Cx32 was probed with mouse anti-Cx32 clone 7C6.6 (1:1200). Signal was detected with Alexa-Fluor-conjugated secondary antibodies (Molecular Probes) and the coverslips were mounted using ProLong Gold plus DAPI (Molecular Probes) or Fluoromount (Invitrogen) with DAPI.
For proliferation studies, cultures were treated with BrdU (10 μM, Molecular Probes) overnight and fixed with Histochoice as described. Immunofluorescent detection of Cx32 preceded denaturation and immunolabeling for BrdU. Following immunolabeling for Cx32, samples were denatured 30 min with 2 N HCl in 0.1% Triton-X-100, neutralized with 0.2 M Borate buffer, pH 9.0 and washed with PBS. The samples were then processed for BrdU incorporation using monoclonal anti-BrdU (LabVision/ThermoFisher;1:150) immunofluorescence.
For proliferation studies, cultures were treated with BrdU (10 μM, Molecular Probes) overnight and fixed with Histochoice as described. Immunofluorescent detection of Cx32 preceded denaturation and immunolabeling for BrdU. Following immunolabeling for Cx32, samples were denatured 30 min with 2 N HCl in 0.1% Triton-X-100, neutralized with 0.2 M Borate buffer, pH 9.0 and washed with PBS. The samples were then processed for BrdU incorporation using monoclonal anti-BrdU (LabVision/ThermoFisher;1:150) immunofluorescence.
8
Immunofluorescence Staining of GFP Fusion Proteins
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HeLa cells were transfected overnight with GFP fusion proteins using FuGene 6 transfection reagent (Roche). Cells were fixed in 4% paraformaldehyde (PFA) for 15 min, washed in PBS, and permeabilized in 0.25% Triton X-100 for 3 min at room temperature or in 100% methanol for 10 min at −20°C when using Tom20 antibody. Cells were stained with organelle-specific antibodies for 1 h at room temperature. Antibodies were visualized by secondary antibodies conjugated to Alexa Fluor 594. Cells were mounted in ProLong Gold plus DAPI (catalog no. P36931; Invitrogen), and z-stacks were collected on an AxioImager M2 microscope (Zeiss).
9
Wound Healing and Invasion Assays
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For wound healing, cells were grown to confluence on glass coverslips. Cells were manually wounded with a pipet tip. 24 hours later, cells were fixed and mounted onto slides with Prolong Gold plus DAPI (Invitrogen). Images were taken with a Leica DMLB microscope equipped with a Diagnostic Instruments Color Mosaic camera and software. Invasion assays were performed as described24 (link).
10
Immunofluorescent Staining of Ear Skin
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Ear skin samples were fixed overnight at 4° C with 1% paraformaldehyde, washed with PBS (6 × 15 min), and then blocked overnight with PBS + 0.3% TX-100 + 20% Aquablock. Next, samples were incubated overnight with a goat anti–Lyve-1 antibody (1:1,000; AF2125; R&D Systems). Following the overnight incubation, samples were washed with PBS + 0.3% TX-100 (3 × 40 min), incubated overnight with an FITC-conjugated anti-goat antibody (1:500), and then washed with PBS + 0.3% TX-100 (3 × 40 min). Samples were mounted with ProLong Gold plus DAPI (P36935; Invitrogen).
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