Immunofluorescence stainings were performed on undifferentiated and adipocyte-differentiated ASC at 1, 2, and 3 wk under adipogenic differentiation. Cells were fixed for 30 min in 3% paraformaldehyde, permabilized for 15 min with 0.1% saponin (Sigma), blocked for 15 min in phosphate-bufferend saline (PBS) with 0.01% Tween-20 (PBST) with 3% (fatty acid free) BSA and 0.1% saponin (PBST-BSA-SAP) (±3 min incubation with 6 M guanidine HCl (Sigma) in 50 mM Tris, pH = 7.5), and washed four times in PBS before reblocking in PBST-BSA-SAP for 30 min. Primary antibody solution containing the indicated antibody or Nile Red (as described in Fink et al., 2004 (link)) were prepared in PBST-BSA-SAP incubated over night at room temperature. Coverslips were washed three times in PBST-BSA-SAP before incubation in secondary Alexa fluor 488 donkey anti-mouse (1:500) (Invitrogen), Alexa fluor 546 donkey anti-rabbit (1:500) (Invitrogen), and/or Alexa fluor 488 anti-rabbit (1:500) (Invitrogen) antibodies for 5 h. Coverslips were washed three times for 5 min in PBS with 0.1% saponin, incubated 5 min with 4’,6-diamidino-2-phenylindole (DAPI) (Sigma), rinsed in dH2O, and mounted with Fluromount G. Images were acquired with an LSM510 META confocal microscope (Zeiss, Oberkochen, Germany) fitted with a 100× NA 1.45 oil plan Apochromat objective and using Zen 2009 software.
Saponin
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Japan, France, Sao Tome and Principe, Panama, Canada, Denmark, Ireland, Netherlands, Switzerland
Saponin is a natural, plant-derived compound that possesses surfactant properties. It can be used as a laboratory reagent for various applications, such as cell lysis, protein extraction, and assay development.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (132 mentions)
Paraformaldehyde (pfa), by Merck Group (100 mentions)
Brefeldin a, by Merck Group (68 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (47 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (42 mentions)
Ionomycin, by Merck Group (41 mentions)
Paraformaldehyde (pfa), by Electron Microscopy Sciences (39 mentions)
Facscanto 2, by BD (35 mentions)
Facscalibur, by BD (32 mentions)
Triton x 100, by Merck Group (32 mentions)
Hoechst 33342, by Thermo Fisher Scientific (32 mentions)
Lsm 710 confocal microscope, by Zeiss (28 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (28 mentions)
Saponin, by Thermo Fisher Scientific (21 mentions)
Lsm 780 confocal microscope, by Zeiss (21 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (21 mentions)
Phorbol myristate acetate (pma), by Merck Group (20 mentions)
Lsrii flow cytometer, by BD (20 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (20 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (19 mentions)
1 217 protocols using saponin
1
Immunofluorescence Analysis of Adipocyte Differentiation
2
Immunofluorescence Staining of Type-II Cells
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Cells grown on 12 mm coverslips were fixed using 3.8% paraformaldehyde (Sigma Aldrich), permeabilized by PBS supplemented with 0.05% Saponin (Sigma Aldrich), blocked using 5% goat serum, incubated with specified primary antibody, diluted as indicated for each antibody, in PBS supplemented with 0.05% Saponin for 2 h at RT, washed with PBS supplemented with 0.05% Saponin three times, and incubated with an appropriate secondary antibody. Hoechst (Sigma-Aldrich) staining was used to visualize nuclei. The primary antibodies used to detect Type-II cells were anti-ATP-binding cassette subfamily A member 3 antibody [3C9] (Abcam, Cambridge, UK, 1:100) and secondary antibody goat anti-Mouse IgG Alexa Fluor 594 594 (Molecular Probes, Cat. # A11032, 1:500). Cells were mounted using Gelvatol mounting media (10.1101/pdb.rec10252 Cold Spring Harb Protoc 2006).
3
Immunohistochemistry of Murine Pinnae
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Freshly recovered pinnae were fixed in PBS/4% PFA on ice for 30 min. Pinnae were then rinsed with cold PBS and transferred to PBS/15% sucrose for a further 1 h on ice. Fixed pinnae were then embedded in optimal cutting temperature (OCT) medium (Sakura Finetek, Netherlands), and frozen at −80 °C overnight. Frozen pinnae were equilibrated to −20 °C and then cut into 5 μm sections which were affixed to glass microscope slides at room temperature for at least 18 h. All subsequent processing was conducted at room temperature. Sections were simultaneously blocked and permeabilised in PBS/5% goat serum/0.05% saponin (Sigma, UK) for 30 min and then incubated with primary antibodies diluted in PBS/5% goat serum/0.05% saponin for 1 h. After three washes in PBS/0.05% saponin, sections were incubated with fluorophore-conjugated secondary antibodies for 45 min without light exposure (primary and secondary antibodies are listed in Supplementary Table S1 ). Finally, sections were washed three times in PBS/0.05% saponin, counter-stained with 2 μg/ml of DAPI (Life Technologies) for 5 min and washed two further times in distilled water. Slides were mounted in Prolong Gold AntiFade reagent (Life Technologies) overnight prior to analysis using a Zeiss 710 inverted confocal microscope. All grouped images were analysed using identical acquisition settings in Zeiss ZEN software.
4
Cardiac Differentiation Efficiency Analysis
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The efficiency of cardiac differentiation was evaluated using flow cytometry. At day 15–20 of the differentiation, cells were washed with PBS, dissociated using 0.25% trypsin for 10 minutes at 37°C, and then fixed in 4% PFA for 10 minutes at room temperature. Subsequent to two washing steps in FACS buffer solution containing 0.5% w/v of Saponin (Sigma-Aldrich), 5% FBS, 1% BSA (Sigma-Aldrich) in 1× PBS, the cells were incubated for 45 minutes with primary antibodies in FACS buffer solution w/Saponin, washed twice in FACS buffer w/Saponin, and finally incubated for 45 minutes with secondary antibodies. For flow analysis, cells were washed 2× in FACS buffer w/Saponin and resuspended in 1× PBS. All experiments included the appropriate iso-type controls. At least three independent experiments were performed. The following primary and secondary antibodies were used: anti-CD90 Thy1 (mouse monoclonal, 1:250, abcam), Anti-Troponin T Cardiac isoform Ab-1 (mouse monoclonal, 1:100, thermo), Anti-CD31 (rabbit monoclonal, 1:100, abcam), Anti-Calponin (rabbit monoclonal, 1:200, abcam), and Alexa488 (goat-anti-mouse or goat - anti - rabbit, 1:200, Invitrogen). Analysis was performed on the LSR Fortessa Analyzer in the flow cytometry facility at UC Berkeley.
5
DNA Extraction from Dried Blood Spots
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DNA extraction was carried out using the Chelex extraction method as previously described [31 (link)] with minor modifications. Briefly, two discs of the dried filter paper blood-spots (DBS) were punched using a 3 mm paper punch into a 1.5 ml microcentrifuge tube containing 1120 μl of 0.5% saponin (0.5 g saponin (Sigma-Aldrich, USA) in 100 ml phosphate buffered saline (Sigma-Aldrich, USA), pH 7.4 (PBS) solution. The tubes were vortexed and then left shaking on a shaking incubator overnight at room temperature. The saponin solution was subsequently decanted, the discs washed twice with 1 ml of ice-cold PBS and centrifuged for 10 min at 10,000×g. A 150 μl aliquot of 6% Chelex-100 (Sigma-Aldrich, USA) in DNase/RNase-free water was added to each disc. After a 5-min incubation at 95 °C the tubes were centrifuged at 14,000×g and the DNase/RNase-free water containing the extracted genomic DNA carefully pipetted into a labelled tube and stored at − 20 °C until further use.
6
Immunofluorescent Staining of Fibronectin and HA
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For staining of fibronectin or HA alone, methanol-fixation was used, whereas in experiments requiring actin and integrin staining, paraformaldehyde (PFA) fixation was used. Briefly, the cell layers were washed with PBS, fixed for 10 min with ice cold methanol or for 20 min with 4% PFA (Thermo Scientific), and then permeabilized with 0.5% saponin (Riedel-de Hae ¨n, Seelze, Germany, #70940) for 10 min. Subsequently, after blocking for 1 h with 3% bovine serum albumin (BSA, Sigma-Aldrich, #A7906; in 0.1% saponin-PBS for PFA-fixed samples), the cultures were first incubated overnight at 4 1C with the primary antibodies and HABP in 1% BSA (and 0.1% saponin for PFA-fixed samples), washed, and then followed by secondary antibodies or other dyes (such as DAPI and phalloidin) for 2 h at room temperature. Finally, the samples were washed with PBS mounted in Prolong Glass (Invitrogen, Life Technologies, Eugene, Oregon, USA; #P36984), and visualized.
7
Phytochemical Interventions in ICR Mice
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After stabilization, the ICR mice were randomly assigned to a control group (n = 8), three groups treated with phytochemicals including Curcumin (n = 8), quercetin (n = 8), and saponin (n = 8), respectively. A positive control group was also established (n = 8). Curcumin, quercetin, and saponin were orally administered daily at a dose of 50 mg/kg body weight regularly. In the positive control group, perindopril was orally administered daily at a dose of 1 mg/kg body weight, under similar conditions as the phytochemical-treated groups. The test materials, quercetin (95%), saponin (8–25%), Curcumin (Curcuma longa L., 65%) and perindopril were purchased from Sigma (Sigma Aldrich, St. Louis, MO, USA). Curcumin, quercetin, saponin and perindopril were dissolved in 0.1% Tween 80 (Sigma-Aldrich Co., St. Louis, MO, USA) according to the daily dose immediately prior to administration. The control group was treated with the same amount of physiological saline.
8
Immunocytochemistry of Induced Pluripotent Stem Cells
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NPC iPSCs were dissociated with dispase and plated to MEF or Matrigel coated chamber slides containing hESC media or mTeSR1, respectively. Cells were cultured for 4 d then processed for immunocytochemistry. Chambers were fixed with 4% PFA for 30 min, PBS rinsed, permeabilized with 0.2% saponin (Sigma) in 10% goat serum, PBS rinsed, and incubated overnight at 4 °C with 50 μg/mL freshly prepared filipin (Sigma) and rabbit anti-OCT4A diluted in 0.05% saponin/10% goat serum. Cells were rinsed and primary antibody detected with goat anti-rabbit 555 IgG diluted in 0.05% saponin/10% goat serum/50 μg/mL filipin. Slides were mounted, imaged with a Leica DM6000B microscope, and processed with Volocity (PerkinElmer) and Adobe Photoshop (Adobe Systems) software.
9
Visualizing Autophagy in BCG-Infected Macrophages
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Differentiated THP-1 cells were seeded onto a poly-D-lysine coated, 96-well glass-bottom black tissue culture plate (4.5 × 104 cells/well) and kept in RPMI-1640 medium minus phenol red (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS at 37°C/5% CO2. Cells were infected with BCG at a MOI of 10, with/without 100 nM 7α,25-OHC, with/without 10 µM GSK682753 for 2 h, washed and incubated for a further 4 h with agonists and antagonists. Cells were then fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0.05% saponin (Sigma Aldrich) for 20 min and blocked with 1% BSA, 0.05% saponin (Sigma Aldrich) for 1 h. Cells were immunolabeled with rabbit anti-human LC3B (ThermoFisher L10382; 1:1,000), 0.05% saponin at room temperature for 1 h followed by Alexa FluorTM 647 goat anti-rabbit IgG (ThermoFisher A21245; 1:1,000), 0.05% saponin at room temperature for 1 h followed by nuclear staining with Hoechst 33342 (Thermo Fisher Scientific 62249; 1:2,000) for 15 min. Cells were washed, and confocal microscopy was performed using the Olympus FV3000, 60× magnification. Images obtained were analyzed with the ImageJ software (24 (link)).
10
Visualizing Autophagy in Neuroblastoma Cells
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Cells were seeded in 24-well plates (8 × 104 cells/well for SK-N-BE(2); 6 × 104 cells/well for LA1-5s), on slide covers coated with 40 μg/ml poly-L-lysine (Sigma-Aldrich). After 24 h, cells were treated with the indicated treatments for 12 h. Cells were fixed with 4% paraformaldehyde (Sigma-Aldrich), permeabilized with 0.02% saponin (Sigma-Aldrich) and blocked for 1 h with 0.01% saponin, 10 mM glycine and 5% BSA (bovine serum albumin; Sigma-Aldrich). Next, cells were incubated overnight with the LC3 primary antibody diluted in 0.01% saponin and 1% BSA in a wet chamber at 4 °C, and then incubated for 1 h with the secondary antibody. Nuclei were stained with Hoechst 33342 (Sigma-Aldrich) for 5 min, rinsed and mounted on a slide with a drop of Fluorsave mounting medium (Calbiochem, Darmstadt, Germany). Images were captured using a fluorescent microscope (Nikon Eclipse 90i, Nikon, Vienna, Austria).
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