Synovial tissues were serially cut into sections and embedded with paraffin using microscope cover glasses. For immunofluorescent staining, tissue sections were deparaffinised and rehydrated with xylene and ethanol. The sections were immersed in antigen retrieval solution (Agilent, Santa Clara, CA, USA) citrate buffer (pH 6) and incubated in a microwave for 20 min. The slides were allowed to cool at room temperature for 30 min before washing with DPBS. Next, the slides were blocked with 1% BSA in TBS+ 0.2% Triton X‐100 (Bio Basic). The sectioned tissues were stained with the following cocktails of primary antibodies in blocking buffer overnight at 4°C: rat anti‐CD8 (1:100), mouse anti‐CD69 (1:100) and rabbit anti‐CD103 (1:100); mouse anti‐IL‐15 (1:400), goat anti‐IL‐15Rα (1:400), rabbit anti‐CD55/DAF (1:50) and/or rabbit anti‐CD68 (1:50); or rat anti‐CD8 (1:100), mouse anti‐perforin (1:50) and rabbit anti‐histone H3 (R2 + R7 + R18)(1:50). Subsequently, the sections were washed and stained for 1 h at room temperature with the following donkey‐raised secondary antibodies: anti‐mouse Alexa Fluor 647 (1:500), anti‐goat Cy3/Cy5 (1:500), anti‐rabbit FITC (1:500) and anti‐rat Cy3/Cy5 (1:500). After washing, sections were counterstained with Hoechst 33342 (Invitrogen) and mounted using ProLong Gold Antifade Mountant (Invitrogen).
Triton x 100
Manufactured by Bio Basic
Sourced in Canada
Triton X-100 is a non-ionic detergent commonly used in biochemistry and molecular biology applications. It is a clear, viscous liquid that functions as a surfactant, helping to solubilize and extract proteins and other biomolecules from biological samples.
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Lab products found in correlation
Paraformaldehyde (pfa), by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Capricorn (2 mentions)
Ethylene diamine tetraacetic acid (edta), by Thermo Fisher Scientific (2 mentions)
Rat tail collagen 1, by Corning (2 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
12 well culture plate, by Corning (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Antigen retrieval solution, by Agilent Technologies (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Hoechst 33342, by Thermo Fisher Scientific (1 mentions)
Ammonium persulphate, by Merck Group (1 mentions)
Bis acrylamide, by Merck Group (1 mentions)
Isoniazid, by Merck Group (1 mentions)
Pyrazinamide, by Merck Group (1 mentions)
Temed, by HiMedia (1 mentions)
Mmp 9, by Merck Group (1 mentions)
Sauton s media, by HiMedia (1 mentions)
19 protocols using triton x 100
1
Immunofluorescent Analysis of Synovial Tissue
2
Mycobacterial Matrix Metalloproteinase Assay
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Isoniazid, rifampicin, pyrazinamide, dexamethasone, MMP-9, SB-3CT and gelatin were obtained from Sigma (St. Louis, MO, USA). Oleic albumin dextrose catalase (OADC) enrichment and 7H11 agar were from Difco-Becton-Dickinson (Becton, Dickinson & Company, MD, USA); Sauton's media was obtained from HiMedia Laboratories (HiMedia Laboratories Pvt. Ltd., Mumbai, India). Acrylamide, bisAcrylamide, ammonium persulphate (Sigma, St. Louis, MO, USA), TEMED (HiMedia Laboratories Pvt. Ltd., India), Triton X-100 (BIO BASIC Inc., Canada), Brij-35 (LOBA CHEMIE Pvt. Ltd., (India)) and other reagents used in zymography were of molecular grade. Ultrapure water was used throughout the study.
3
Purification and Characterization of Enzymes
4
Immunocytochemical Analysis of Autophagy and Dopaminergic Markers
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After fixation with 4% paraformaldehyde, SH-SY5Y cells were blocked with 0.5% Triton X-100 (Biobasic Inc., Shanghai, China) supplemented with 10% goat serum (Biobasic Inc.) for 30 minutes and then incubated with primary antibody LC3B (1:500), p62 (1:500), and TH (mouse, 1:250, Cat# 66334-1-Ig, RRID: AB_2881714, Proteintech) overnight at 4°C. Next, the cells were covered with fluorescent antibodies at room temperature for 2 hours: Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L) (1:1000, Cat# A0423, RRID: AB_10698242, AB_2891323, Beyotime) and Alexa Fluor 647-conjugated goat anti-mouse IgG (H+L) (1:1000, Cat# A0473, RRID: AB_2891322, Beyotime). After washing with phosphate buffered saline-Tween 20, the nuclei were stained with Hoechst 33342. Images were captured using a fluorescence microscope (Olympus).
5
Immunohistochemical Analysis of Neurogenesis
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Scopolamine hydrobromide, 5′-Bromo-2′-deoxyuridine (BrdU), and 4% parafor-maldehyde (PFA) solution were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). 2′-7′-Dichloro-fluorescin diacetate (DCFDA) was purchased from Invitrogen Molecular Probes (Eugene, Oregon, USA). Triton X-100 and 3% goat serum were purchased from Biobasic (Biobasic Inc., Ontario, Canada) and Gibco (Gibco BRL, Grand Island, NY, USA), respectively. Staining for immunohistochemistry, primary antibodies against BrdU, doublecortin (DCX) and neuronal nuclei (NeuN) were obtained from Abcam (Abcam, Cambridge, MA, USA), Cell Signaling Technology (Danvers, MA, USA)and Millipore (Millipore, Billerica, MA, USA), respectively. Anti-phosphorylated (p) Akt (ser 473), total Akt, pCREB (ser 133), total CREB were purchased from Cell Signaling Technology. Anti-β-actin was obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Secondary antibodies for immunostaining were purchased from Vector Laboratories (Vector Laboratories Inc., CA, USA) or Molecular Probes (Eugene, OR, USA). Secondary antibodies for Western blotting were obtained from Thermo Scientific (Thermo Scientific, MA, USA).
6
Synthesis and Characterization of Iron-Based Nanoparticles
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FeCl2·4H2O (p.a.) and FeCl3·6H2O (99.7 %) salts were purchased from Anedra (Research-AG) and Supelco (Merck), respectively. Tetraethyl orthosilicate (TEOS, 99 %) was acquired from Sigma-Aldrich. Ammonium hydroxide (NH4OH, 25 % wt) was obtained from Ciccarelli. Absolute ethanol (≥99.5 % V/V), EDTA (ethylenediaminetetraacetate, p.a) and isopropanol (catalog number: 2000971600) was purchased from Biopack. Guanidinium thiocyanate (catalog number: G9277) for molecular biology with a purity of ≥99 % was purchased from Sigma-Aldrich. Triton X-100 (catalog number: TB0198), of biotech grade, was acquired from BioBasic. Tris HCl buffer was prepared using Tris (Tris-Hydroxymethyl-aminomethane, catalog identifier: RU2510) with a purity of ≥99 %, purchased from GenBiotech, and HCl (catalog number: 918, 36.5–38.0 % Pro-analysis) was obtained from Ciccarelli. DTT (DL-Dithiothreitol; Cleland's reagent) was purchased from Thermo Scientific. Trypsin and Minimum Essential Medium (MEM) from Gibco™ and fetal bovine serum (code: FBI) from Internegocios S.A. were used for assays involving A549 cells. All chemicals were used as received. Experiments were conducted using ultrapure water (EMD Millipore, USA).
7
Quantitative Analysis of Xio1-GFP Protein
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For Western blot analyses, total proteins were extracted from rice or Arabidopsis plants using a protein extraction buffer [6.8 mM Na2HPO4 (Biobasic), 3.2 mM NaH2PO4 (Biobasic), 150 mM NaCl (Biobasic), 2 mM EDTA (Biobasic), 200 mM NaF (Biobasic), 1% Triton X-100 (Biobasic), and 1 mM PMSF (Biobasic)]. Protein concentration was determined using a previously described method (Bradford, 1976 (link)). Proteins (100 μg) were loaded per well, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and subsequently blotted onto nitrocellulose membrane (Bio-Rad) for immunodetection. For Xio1-GFP detection, anti-GFP antibody (B-2) SC-9996 (Santa Cruz Biotechnology) and horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody SC-2031, (Santa Cruz Biotechnology) were used as a primary and a secondary antibody at a final dilution of 1:1,000 and 1:10,000 for 2 h, respectively. GFP bands were visualized using the SuperSignal West Pico Chemiluminescent Substrate (Thermo).
8
Immunofluorescence Staining of DNA Damage
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For immunofluorescence staining, cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 10 min at room temperature and permeabilized with 0.5% Triton™ X-100 (Bio Basic). Specific antibodies against 8-OxoG (Abcam), phospho-histone H2AX (Ser139; Millipore), phospho-CHK1 (Ser345), phospho-P53 (Ser15), PARP1, cleaved-caspase 3 (Cell Signaling Technology), and poly-ADP-ribose (Enzo Life Sciences) were used for visualization of the proteins. The images were captured using a fluorescence microscope (Nikon) equipped with the NIS-Elements 4.0 Nikon imaging software. For quantification, a minimum of 500 cells were analyzed from each of three independent experiments.
9
Optimized Protein Extraction Protocol
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The lysates in Fig. 1D were prepared with the 2 × sodium dodecyl sulfate (SDS; Affymetrix/USB, J18220) buffer in the GFP-Trap®_A protocol (ver. 2014-12-18) offered by ChromoTek (Planegg-Martinsried, Germany). The rest of the lysates were prepared with 1% (v/v or w/v) of the indicated detergents in the dilution buffer for GFP-Trap®_A, with cOmplete™ EDTA-free Protease Inhibitor Cocktail (Roche, 04693159001) instead of the suggested protease inhibitors. The detergents used were Tween 20 (Biosesang, T1027), Triton X-100 (Bio Basic, TB0198), Nonidet P-40 (Bio Basic, NDB0385), Octyl-β-D-glucopyranoside (Fluka, 75083), n-Dodecyl β-D-maltoside (DDM; ThermoFisher, 89903), CHAPS (Sigma-Aldrich, C5070), CHAPSO (Calbiochem, 220201), and Zwittergent 3–16 (Calbiochem, 693023). 300 μl of lysis buffer was added per tube and the tube was vortexed for 1 min to produce a homogenous mixture. The mixture was then centrifuged for 5 min at 10,000 × g at 4°C (10°C for Zwittergent 3–16- or SDS-solubilized lysates) to remove tissue debris. The intermediate supernatant was transferred to a fresh tube and centrifuged once more to obtain the final transparent tissue lysate. Total protein quantification was performed with a Pierce™ BCA Protein Assay Kit (ThermoFisher, 23227). The final lysates were stored at −80°C.
10
Immunofluorescence Staining for Cell Fixation and BrdU Detection
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NPC or OPC monolayer cultures were fixed with 4% PFA for 10 min at room temperature. The cells were then permeabilized with 0.2% NP-40 in 1x PBS, blocked with 0.5% BSA, 6% normal donkey serum (Jackson ImmunoResearch) in 1x PBS, and incubated with primary antibodies (listed below) in 1⁄2 blocking buffer diluted with 1x PBS for 2h at room temperature or overnight at 4°C. After extensive washing, appropriate secondary antibodies (listed below) were added in 1x PBS for 1h at room temperature. At this point, nuclei were stained using Hoechst solution (Riodel-De Haen Ag) and coverslips were mounted in Fluoromount-G (Invitrogen), or cells were further processed for BrdU staining. Here, cultures were post-fixed with 4% PFA and then incubated with 1M HCl at 4°C for 10 min, followed by 2M HCl for 20 min at room temperature. The cells were then blocked with 1M glycine (Sigma), 1% Triton X-100 (Bio Basic), and 5% normal donkey serum (Jackson ImmunoResearch) for 30 min at room temperature. After blocking, anti-BrdU (Abcam, 1/1000 dilution) was added to the cells for 1h at room temperature or at 4°C overnight. Secondary antibodies (Donkey anti-sheep-647, Jackson ImmunoResearch, 1/500) were then added to the cells for 1h. Nuclei were stained using Hoechst and coverslips were mounted as described above.
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