The MCF-7/WT and MCF-7/DOX-1 cells were fixed onto glass coverslips by 4% paraformaldehyde (P6148, Merck, Germany) for 10 min, and subsequently stained with either monoclonal antibody anti-CD24 conjugated to Milli-Mark™ fluorescein isothiocyanate (FCMAB188F, anti-CD24–FITC; clone SN3, 1:15 (v/v) dilution, EMD Millipore Corporation, Burlington, MA, USA) or monoclonal antibody anti-human CD44 conjugated to R-phycoerythrin (CBL154P, anti-CD44–R-PE; clone F10-44-2, dilution 1:15, EMD Millipore Corporation, Burlington, MA, USA) at room temperature (RT) for 1 h. After washing with PBS (524650, Merck, Germany) containing 0.05% Tween-20 (P9416, Merck, Germany), the cell nuclei were stained with Hoechst 34580 (H21486, 2 µg/mL, Thermo Fisher Scientific, USA) for 10 min. The cells were visualized using a confocal microscope Zeiss LSM 5 DUO (Zeiss, Germany) at λexc = 488 nm and λem = 515–550 nm for anti-CD24-FITC, at λexc = 560nm and λem > 573 nm for anti-CD44-R-PE, and at λexc = 405 nm and λem = 420–480 nm for Hoechst 34580. The photomicrographs were scanned using a 63× objective.
P9416
Manufactured by Merck Group
Sourced in United States
P9416 is a laboratory equipment product offered by Merck Group. It is designed to perform a core function, but a detailed description cannot be provided while maintaining an unbiased and factual approach without extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
T8787, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
L3224, by Thermo Fisher Scientific (3 mentions)
Imagequant las 4000, by GE Healthcare (2 mentions)
Triton x 100, by Merck Group (2 mentions)
D1306, by Thermo Fisher Scientific (2 mentions)
A9647, by Merck Group (2 mentions)
Ab6789, by Abcam (2 mentions)
Supersignal west pico chemiluminiscent substrate, by Thermo Fisher Scientific (2 mentions)
Ab6721, by Abcam (2 mentions)
Ripa lysis buffer, by Merck Group (2 mentions)
Image lab software, by Bio-Rad (2 mentions)
Chemidoc xr, by Bio-Rad (2 mentions)
A8010, by Solarbio (2 mentions)
Ab71916, by Abcam (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
P6148, by Merck Group (1 mentions)
Hoechst 34580, by Thermo Fisher Scientific (1 mentions)
Lsm 5 duo, by Zeiss (1 mentions)
Hoechst 34580, by Zeiss (1 mentions)
43 protocols using p9416
1
Immunofluorescence Analysis of CD24 and CD44
2
Immunocapture of CD9-Enriched Extracellular Vesicles
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CD9-EVs were immunocaptured from CSF and serum samples using plates which contained covalently attached antibodies against the tetraspanin CD9 (HBM-POS-CC/T1, Hansa-BioMed). 100 μl of CSF or serum was loaded per well and incubated in agitation for the first 20 min at room temperature and then overnight at 4 °C. The next day, each well was washed four times with 0.05% Tween 20 (P9416, Merck) PBS (21-031-CV, Corning), solubilised with 50 μl of DNA/RNA/protein solubilisation reagent 100ST (DCQ100ST, DirectQuant), incubated at 90 °C for 3 min with agitation at 750 rpm, and stored at −20 °C until the dPCR assay was performed.
3
Sirt-1 Protein Quantification in Cell Extracts
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During the mRNA extraction, protein samples were collected after bromochloropropane (B9673; Sigma-Aldrich) separation. Ethanol was added and samples centrifuged at 12.000g for 5 min. This procedure was repeated twice. The resulting pellet was suspended in 4% sodium dodecyl sulfate (SDS) (436143; Sigma-Aldrich). Protein content was measured using the Bradford method. Equal amounts of protein (20 μg) were loaded on 10% SDS-polyacrylamide gels, submitted to SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Hercules, CA). Membranes were blocked by 5% non-fat-dried milk (M7409; Sigma-Aldrich) in 50 mM Tris-buffered saline (TBS) containing 0.1% Tween (P9416; Sigma-Aldrich) for 1 h at room temperature, washed, and incubated overnight at 4 °C with monoclonal rabbit anti-Sirt-1 (1:2000; ab32441; Abcam) and polyclonal rabbit anti-β-actin (1:10000; 4967; Cell Signaling, Frankfurt, Germany) antibodies. The next day, membranes were washed and incubated with secondary donkey anti-rabbit antibody. Blots were revealed using a chemiluminescence kit and scanned using a myECL Imager (Thermo Fisher Scientific). Sirt-1 abundance was densitometrically evaluated in three independent experiments. The relative abundance of Sirt-1 was normalized to protein loading as determined in β-actin blots.
4
Protein Extraction and Western Blot Protocol
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Organoids were harvested in Cell Recovery Solution (Corning Cell Recovery Solution, 100 mL #354253) and incubated with rotation for 1 hour at 4°C. Cells were then pelleted, and lysed in 0.1% Triton X-100, 15 mmol/L NaCl, 0.5 mmol/L EDTA, 5 mmol/L Tris, pH 7.5 supplemented with protease Mini-complete protease inhibitors (Roche) and a phosphatase inhibitor cocktail (PhosSTOP; Roche). Cells were incubated on ice for 30 minutes. Protein lysates (10 μg of protein) were separated by SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Bio-Rad #1704274), blocked with 5% BSA in TBST (1% Tween 20; Sigma-Aldrich #P9416; TBS), and incubated with primary antibodies overnight at 4°C. Proteins were detected using HRP-conjugated secondary antibodies (Thermo Fisher Scientific, #A15999: ABCAM #ab6721).
5
Primer Grafting on Flow Cell
6
Western Blot Protein Extraction and Analysis
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Cells were washed with ice-cold phosphate-buffered saline (PBS) and lysed with Pierce RIPA Buffer (89900; Thermo Fisher Scientific), 1 mM DTT (R0861; Thermo Fisher Scientific), and 1* cOmplete Mini EDTA-free Protease Inhibitor Cocktail (11836170001; Sigma-Aldrich). Cells were lysed on ice for 20 min (vortexed every few minutes) and then clarified by centrifugation (13,000 RPM for 10 min). 4× Nu-PAGE sample buffer (NP0007; Thermo Fisher Scientific) was added to lysates to a final concentration of 1×, samples were boiled for 5 min at 95°C, and then loaded into 4–12% Bis-Tris Nu-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% nonfat-dried milk in Tris-buffered saline with 0.1% Tween-20 (P9416; Sigma-Aldrich) (TBST) for 1 h and then incubated with primary antibody 1 h at room temperature or overnight at 4°C. Antibody dilutions are listed in Table S1 . Membranes were washed 3× with TBST and then incubated with secondary antibody at room temperature for 1 h in 5% nonfat-dried milk in TBST. Membranes were washed 3× again in TBST, and antibody detection was achieved by rocking membranes in Pierce ECL Western blotting substrate (32106; Thermo Fisher Scientific) for 5 min.
7
Western Blot Analysis of Transfected Cells
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Cells were seeded in 6-well dishes and transfected as indicated. One day post transfection the cells were lysed in 1xSDS buffer (50mM Tris pH 7.4; 2% SDS; 10% Glycerol) supplemented with 200mM dithiothreitol (Sigma, #D0632) and heated at 100°C for 10 minutes. The lysates were resolved by SDS-PAGE and transferred to nitrocellulose membrane (Sigma, GE10600003). The membrane was stained with Ponceau S (Sigma, P3504), blocked with 5% non-fat dry milk in 1% TBS-T (0.2M Tris pH 8; 1.5M NaCl and 0.05% Tween20 (Sigma, P9416)) and then incubated with indicated primary antibodies for 24h. The membrane was washed three times for 10 minutes each with PBS-T followed by incubation with secondary antibody for 1h. The membrane was washed three times for 10 minutes and analyzed by enhanced chemiluminescence using the ImageQuant LAS 4000 (GE Lifescience).
8
Immunocytochemistry of Neural Cell Markers
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Cells were cultured and differentiated in a 96-well plate covered with poly-L-ornithine hydrobromide (Sigma-Aldrich; P3655) and Laminin (10% in PBS). Cells were grown overnight, after which they were fixed with 4% paraformaldehyde (paraformaldehyde 16%, Alfa Aesar) in PBS for 30 min and permeabilized with permeabilization buffer (0.5% Triton X-100 (Sigma-Aldrich) in PBS). The cells were washed with washing solution (25% BSA (bovine serum albumin), Sigma-Aldrich) and 0.1% Tween-20 (Sigma-Aldrich, P9416; PBS). The cells were blocked for 1 h with 10% goat serum in wash buffer and subsequently in washing solution. The blocking buffer was removed and the cells were labeled with primary antibodies (anti-nestin 1:100; Abcam (10C2); anti-TUBIII 1:75 (Merck (MAB1637); anti-GFAP 1:100 (Santa Cruz (2E1)) diluted in blocking buffer and incubated overnight at 4 °C. The cells were incubated at room temperature (RT) for 30 min and then washed using washing solution. A secondary antibody (ENCO, Alexa 549) was added (1:200 in blocking solution) for 1 h at RT. The cells were washed with the washing solution and stained with DAPI for 1 min. The cells were washed three times with DDW. Images were taken using a fluorescent microscope.
9
Immunostaining and Expansion Microscopy
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Punches were blocked in IF buffer (1% BSA; [Sigma-Aldrich; A9647] and 0.05% Tween-20 [Sigma-Aldrich; P9416] in 1× PBS) for 1–2 h at RT, and incubated with primary antibody diluted in IF buffer for 24 h at 4°C. Punches were washed in 1× PBS for 1–2 h and incubated with secondary antibody and DAPI (1:10,000 final dilution; Thermo Fisher Scientific; D1306) in IF buffer for 24 h at 4°C. The following primary antibodies were used for immunostaining: mouse anti-acetylated tubulin (Sigma-Aldrich; T7451) at 1:4,000, rabbit anti-β tubulin (Abcam; ab15568) at 1:250, rabbit anti-ARL13B (Abcam; ab83879) at 1:500, rabbit anti-Cep290 (Abcam; ab84870) at 1:600, and rabbit anti-Cep164 (Proteintech; 22227–1-AP) at 1:500. Secondary antibodies Alexa Fluor 488 anti-mouse (Thermo Fisher Scientific; A11029) and Alexa Fluor 555 anti-rabbit (Thermo Fisher Scientific; A21429) were used at a 1:800 dilution to label primary antibodies for 24 h. After immunostaining, the samples were expanded in deionized H2O for 2 h at RT with deionized H2O exchanged every 10 min and additionally overnight at 4°C. Prior to imaging, expanded punches were mounted in Rose chambers, Attofluor cell chambers, or glass-bottom Microwell dishes (MatTek; P35G-1.5-14-C).
10
Immunoblot Analysis of Mitochondrial and Autophagy Proteins
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Proteins were separated by SDS–PAGE under reducing conditions and transferred to poly(vinylidene fluoride) membranes. Membranes were blocked with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween 20 (Sigma-Aldrich, P9416; PBST). The blots were incubated overnight at 4 °C with primary antibodies. Immunoblot analysis was performed using an enhanced chemiluminescence procedure (GE Healthcare, RPN2106), and western blot images were captured using the ChemiDoc™MP system (Bio-Rad), Image Lab version 6.1.0. Densitometric analysis was performed using ImageJ software (National Institutes of Health). The following antibodies were used: Cell Signaling Technology: anti-COXIV (3E11, #4850), 1:1000; anti-VDAC1 (D73D12, #4661), 1:1000; anti-Tomm20 (D8T4N, #42406), 1:1000; anti-GAPDH (14C10, #2118), 1:10000; anti-Cytochrome c (136F3, #4280), 1:1000; anti-LC3B (#2775), 1:1000; Novus Biologicals: anti-Dhps (NBP1-82648), 1:1000; BD Biosciences: anti-Eif5a (611976), 1:1000; Sigma-Aldrich: anti-Dohh (HPA041953), 1:1000; Millipore: anti-Hypusine (ABS1064), 1:1000; anti-puromycin (12D10, #MABE343), 1:1000; Abcam: anti-Tfam (ab131607), 1:1000; anti-PGC1α (ab54481), 1:1000; anti-TFEB (ab2636), 1:1000; and Atp5a, Uqcrc2, Sdhb, and Ndufb8 were detected using Total OXPHOS Rodent WB Antibody Cocktail (ab110413), 1:1000. Primary antibodies were diluted in 1% BSA containing PBST.
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