CT26 cells were incubated with FeCl3, TA-Fe3+, DOC, DOC@TA-Fe3+, and DOC@TA-Fe3+ + DFO. After incubation for 24 h, the cells were fixed with 4% paraformaldehyde (Servicebio, Wuhan, China) for 20 min, permeabilized with 0.1% Triton-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 min, and sealed with 1% bovine serum albumin (Aladdin) for 1 h. The cells were then treated with antitubulin antibodies (1:150; HuaBio), incubated with goat antirabbit secondary antibody (HuaBio) for 2 h, and stained with 4′,6-diamidino-2-phenylindole (Aladdin). Finally, the cells were observed with CLSM.
Triton 100
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, China
The Triton-100 is a laboratory instrument designed for performing various analytical tasks. It is a compact and versatile device that can be utilized for a range of applications within the scientific research and testing domains.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (14 mentions)
Tween 20, by Merck Group (7 mentions)
Paraformaldehyde (pfa), by Merck Group (7 mentions)
Hoechst 33342, by Thermo Fisher Scientific (7 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Lsm 510, by Zeiss (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Sodium chloride (nacl), by Merck Group (4 mentions)
Trypsin, by Thermo Fisher Scientific (3 mentions)
B27 supplement, by Thermo Fisher Scientific (3 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Sodium deoxycholate, by Merck Group (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Methanol, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Normal goat serum (ngs), by Abcam (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Agilent Technologies (2 mentions)
Rabbit polyclonal igg, by BioLegend (2 mentions)
Bovine serum albumin (bsa), by BioLegend (2 mentions)
Leica dm6000b fluorescent microscope, by Leica Microsystems (2 mentions)
87 protocols using triton 100
1
Imaging of CT26 cells with iron compounds
2
Immunofluorescence Staining of MSCs
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MSCs were fixed with 4% paraformaldehyde solution (Panreac) at room temperature for 10 min and incubated with 0.2% Triton ×100 (Sigma, St. Louis, MO, USA) solution at RT for 10 min (except neurotrimin labelling). Furthermore, MSCs were incubated for 1 h in 1% bovine serum albumin (BSA, Sigma) and 10% normal goat serum (Abcam, Cambridge, UK) solution at room temperature to block the non-specific interaction of antibodies. Subsequently, the samples were incubated with primary polyclonal rabbit antibody for αSMA (Biolegend, San Diego, CA, USA, 904601), perilipin (Thermo Fisher Scientific, PA1-1051), CHD3 (Cloud-Clone Corp., Wuhan, China, PAA317Mu01), neurotrimin (Affinity Biosciences, Melbourne, Victoria, Australia, DF4245), RDH10 (Affinity Biosciences, DF12105), or rabbit polyclonal IgG (Biolegend, 910801) in 1% BSA solution at +4° overnight. Then, the samples were incubated with fluorescence-labeled goat anti-rabbit or goat anti-mouse (Invitrogen, A-11001) secondary antibodies (A11034, Invitrogen) at room temperature for 1 h. Cell nuclei were labeled with DAPI (DAKO, Glostrup, Denmark). Samples were analyzed with a Leica DM6000B fluorescent microscope equipped with a Leica DFC 360FX camera (Leica Microsystems GmbH, Wetzlar, Germany) using the LasX program. The percentage of CHD3+ MSCs was evaluated in FIJI using IgG-based thresholding.
3
Immunofluorescence Staining of Oocyte and Blastocyst
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Both oocyte and blastocyst samples were fixed in 4% PFA in PBS for 30 min at room temperature, followed by permeabilization and blocking for 1 h in PBS containing 0.1% Triton‐100 (Sigma) and 10% fetal bovine serum (FBS) (Thermo Fisher Scientific). The samples were subsequently incubated with primary antibodies (4 °C, overnight) and Alexa flour 594/488‐conjugated secondary antibodies (room temperature, 30 min), respectively, and counterstained with Hoechst 33342 for 20 min. The samples were finally mounted on glass slides and examined under an LSM 700 laser scanning confocal microscope (Carl Zeiss). Images for different groups of samples in the same experiment were taken under the same parameters. The primary antibodies used for staining of oocyte spindles and SAC were monoclonal anti‐β‐tubulin antibody produced in mouse (catalog no. T4026; 1:500, Sigma) and sheep polyclonal BubR1 antibody (catalog no. ab28193; 1:200, Abcam, USA). Blastocysts were stained with OCT4 (catalog no. ab28193, 1:500, Abcam,) and CDX2 (catalog no. CA 94538, 1:500, BioGenex, USA) for distinguishing the cells of ICM and TE, respectively.
4
Pigment Extraction from Microbial Biomass
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Pigment was extracted from the biomass in accordance with the method described by Bligh and Dyer (Bligh and Dyer, 1959 (link)). First, 1 mL chloroform and 2 mL methanol were added to the wet biomass of cells (0.8 g). Cells were agitated for 12 h in the extraction mixture and subsequently centrifuged (2,000 × g), followed by the addition of 1 mL water and 1 mL chloroform (to separate phases). The chloroform layer was washed three times with 0.1 M NaCl. The bacterial pellet was re-suspended in ice-cold 100 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer (pH 8.0) containing 2% Triton × 100 (Sigma, United States) then lysed by using zirconium beads on a bead beater homogenizer (MP Biomedicals FastPrep-24) for 1 min, five times and stayed in dark place at room temperature for 5 h. The bacterial lysate was centrifuged at 13,000 rpm for 15 min at 4°C.
5
Biochemical Analysis of Oxidative Stress
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DATS was obtained from LKT laboratories (St. Paul, MN, USA). Folin, GSH, hydrogen peroxide (H2O2), dithiothreitol (DTT), phenylmethylsulfonyl fluoride (PMSF), paraformaldehyde (PFA), β-nicotinamide adenine dinucleotide phosphate reduced (NADPH), GR, pepstatin A, aprotinin, leupeptin, Nonidet® P40, Triton® × 100, ethylenediaminetetraacetic acid (EDTA), phosphatases cocktail inhibitor and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). Sodium azide (NaN3) was obtained from Hycel de México (Zapopan, Jalisco, México). Hydroxyethyl piperazineethanesulfonic acid (HEPES) was from MP Biomedicals (Solon, OH, USA). Primary antibodies rabbit anti-GST (110-218; sc-33614) and rabbit anti-matrix metalloproteinase-9 (MMP-9; H-129; sc-10737) were obtained from Santa Cruz Biotechnology (Dallas, TX, USA). The primary antibody rabbit anti-malondialdehyde (MDA; ab6463) was obtained from Abcam (Cambridge, MA, USA). Primary antibodies rabbit anti-SOD1 (ADI-SOD-101-E) and rabbit anti-SOD2 (ADI-SOD-111) were purchased from Enzo Life Science (Farmingdale, NY, USA). TransAM Nrf2 ELISA kit was from Active Motif (Carlsbad, CA, USA) and Universal L Kit SAB-System HRP was from DAKO (Carpinteria, CA, USA). All other reagents were obtained from other known commercial local sources.
6
Immunofluorescence Staining of Cardiac Tissue
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Heart sections were deparaffinized and rehydrated by gradient elution using xylene and ethanol, followed by PBS washes. Antigen retrieval was performed in Tris‐EDTA solution for 20 min at 95 °C. For NMCMs, cells were rinsed with PBS, fixed with 4% paraformaldehyde for 20 min at room temperature. Prepared heart sections and NMCMs were blocked by incubation with PBS containing 10% goat serum (Beyotime Biotechnology, China) and 0.3% Triton‐100 (Sigma‐Aldrich, USA) for 1 h at 37 °C, and incubated with primary antibodies (Table S2 , Supporting Information) overnight at 4 °C. Slides were washed three times and incubated with Alexa Fluro conjugated secondary antibody (Table S2 , Supporting Information) for 1 h at room temperature. Nuclei were stained using 4′,6‐diamidino‐2‐phenylindole for 10 min. Image acquisition was performed using a Zeiss LSM880 confocal microscope and analyzed using ZEN software (Zeiss, Germany).
7
Immunofluorescence Analysis of SP1 in Pancreatic Cancer Cells
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MIA Paca-2 and PANC-1 cells were grown on 4 well chamber slides. The cells were washed three times with PBS after SP1 siRNA treatment, and then fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.1% Triton ×100 (Sigma) for 10 min. After three time washing with PBS, incubate cells with 1% BSA in PBST (PBS + 0.1% Tween20) for 30 min. Polyclonal rabbit antibodies against human SP1 (1:200, Abcam, Cambridge, UK), F-actin (1:200, Abcam, Cambridge, UK) were applied overnight at 4 °C. Washed cells were incubated with Secondary Alexa Fluor 555 anti-rabbit IgG (1:1000, Cell signaling) and Alexa Fluor 488 anti-mouse IgG (1:1000, Cell signaling) for 1 hour at room temperature, washed again. Cells were mounted in DAPI Staining Solution (Abcam, Cambridge, UK), and analyzed using confocal microscopy (LSM510, Carl Zeiss, Jena Germany).
8
Electrospinning Polyacrylonitrile Nanofibers
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Polyacrylonitrile nanofibers were produced by electrospinning using a solution of 10% W/W Polyacrylonitrile (Sigma Aldrich) dissolved in dimethylformamide (DMF, Sigma Aldrich). The electrospinning set-up (IME Medical Electrospinning, NL) was equipped with a rotating drum module located at a distance of 15 cm from the needle. In a classical experiment, a voltage of 20 kV was applied and the rotating speed of the drum was set at 2000 rpm. To tune NFS mechanical properties, multiwalled carbon nanotubes (MWCNTs, Nanocyl, 95% purity) were added in the Polyacrylonitrile solution and stabilized by adding Triton × 100 (Sigma Aldrich) at a weight ratio of 1:50 of MWCNT/Triton. After electrospinning, the NFSs were cross-linked by heat treatment at 250 °C (4 °C/min) during 2 h under air. Pieces of the NFSs were cut and sterilized using classical autoclave treatment before further biological use [7 (link)].
9
Quantification of Phosphoryl-NF-κB p65 Localization
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Phosphoryl-NF-κB p65 localization was quantified via an immunofluorescence technique. Paraformaldehyde (v/v, 1/25) was used for IPEC-J2 cells fixation for 30 min at a temperature of 37 °C. After a PBS wash (0.1 mM, pH7.4), permeabilised in 0.5% Triton (Triton×100, Sigma, Harz Lower Saxony, Germany) for 20 min, and for 20 min blocked with 5% BSA, as well as hatched with the anti-phosphoryl-NF-κB p65 antibody (diluted 1:100) for the whole night at a temperature of 4 °C. After washing with PBS (0.1 mM, pH7.4) for the second time, the secondary antibody was used to incubate the cells for 1 h at room temperature. Coverslips were washed two times via PBS (0.1 mM, pH7.4), and hatched with the goat anti-rabbit IgG antibody for 1 h in the absence of light, and hatched in a DAPI staining solution for 10 min. After that it was washed again in PBS. The fluorescence was monitored using an Olympus-fluoview ver.3.1 viewer (Olympus Corporation, Miyazaki Prefecture, Kyushu, Japan) [38 (link)].
10
Immunofluorescence Analysis of Retinal Cell Apoptosis
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For Bcl-2, cleaved caspase-3 (Asp175), and 4′,6-diamidino-2-phenylindole (DAPI) staining 1 and 3 days after ischemia, the eyes were quickly enucleated and dissected, and the posterior eyecups were placed in a chilled fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) for 6 hours. After washing three times, the fixed retinas were transferred into 30% sucrose containing 0.1 M PBS overnight at 4°C and then embedded with Tissue-Tek® O.C.T Compound (Sakura Finetek; Tokyo, Japan) at optimal cutting temperature. Cryostat sections of the retina (12 μm, sagittal) were mounted onto poly-L-lysine-coated slides (VWR International, Radnor, PA, USA). The specimens were blocked in 2% horse serum and 2% BSA in Triton 100 (Sigma-Aldrich) for 60 minutes. The Bcl-2 and cleaved caspase-3 (Asp175) antibodies (1:500) were incubated overnight at 4°C. After three 5-minute rinses in PBS, Dylight488 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) secondary antibody (1:1,000) and DAPI (1:50,000) were added and incubated at room temperature in the dark. After three 5-minute rinses in PBS, the slides were covered with Vectashield Mounting Medium (Sigma-Aldrich). Fluorescence signals were visualized by laser-scanning confocal microscopy (Olympus Corporation, Tokyo, Japan).
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