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).
Anti mouse igg alexa fluor 488
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-mouse IgG Alexa Fluor 488 is a secondary antibody conjugate used for detecting and visualizing mouse primary antibodies in various immunoassay applications. The antibody is labeled with the Alexa Fluor 488 fluorescent dye, which has an excitation maximum at 495 nm and an emission maximum at 519 nm.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit igg alexa fluor 555, by Cell Signaling Technology (4 mentions)
Hoechst 33342, by Thermo Fisher Scientific (3 mentions)
Alexa fluor 594 anti rabbit igg, by Cell Signaling Technology (2 mentions)
Paraformaldehyde (pfa), by Thermo Fisher Scientific (2 mentions)
Goat serum, by Avantor (2 mentions)
Anti ha tag 6e2 mouse monoclonal antibody, by Cell Signaling Technology (2 mentions)
Anti gfp d5.1 rabbit monoclonal antibody, by Cell Signaling Technology (2 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (2 mentions)
Lsm 510, by Zeiss (1 mentions)
Triton 100, by Merck Group (1 mentions)
F actin, by Abcam (1 mentions)
Dapi staining solution, by Abcam (1 mentions)
Vector h 1000, by Vector Laboratories (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Lsm 700 confocal module, by Zeiss (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg, by Cell Signaling Technology (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Hela cell, by American Type Culture Collection (1 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Propidium iodide, by Merck Group (1 mentions)
12 protocols using anti mouse igg alexa fluor 488
1
Immunofluorescence Analysis of SP1 in Pancreatic Cancer Cells
2
Immunofluorescence Staining of FoxP3 and GrB
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Antigen retrieval was performed on deparaffinized sections in 10 mM citrate buffer (pH 6.0) in a pressure cooker for 15 min, followed by a wash in 0.1 M phosphate buffer (PB; pH 7.0). Subsequently, to quench any autofluorescence, the sections were treated with 50 mM glycine for 40 min at room temperature. Thereafter, non-specific staining was blocked with 1% bovine serum albumin for 30 min, followed by an overnight incubation at 4 °C with the primary antibodies against FoxP3 (Abcam, Cambridge, UK) and GrB (both antibodies at a 1:90 dilutions). Next, the sections were washed and incubated for 1 h with the secondary antibodies (1:300, Alexa Fluor 594 anti-rabbit IgG, Cell Signaling and 1:300 Alexa Fluor 488 anti-mouse IgG, Cell Signaling). Nuclei were counterstained with DAPI (1 μg/mL, Sigma-Aldrich, St. Louis, MO, USA). Finally, the sections were mounted in Vectashield (Vector H-1000, Vector). The samples were imaged with a Zeiss Axio Observer inverted microscope (×20 or ×40 NA 1.3 oil objectives) equipped with a Zeiss LSM 700 confocal module (Carl Zeiss Microimaging GmbH, Jena, Germany).
3
Investigating Retinoblastoma Cell Therapies
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The WERI-Rb1 human retinoblastoma cell line and HeLa cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Sprague Dawley (SD) rats were obtained from the animal center of Zhongshan Ophthalmic Center, Sun Yat-sen University. TMP, AMD3100, DMSO and propidium iodide (PI) were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Rabbit anti-CXCR4 and GAPDH, mouse anti-microtubule associated protein-2 (MAP-2) primary polyclonal antibodies were purchased from Abcam (Cambridge, UK; cat. no. ab2047), ProteinTech Group, Inc. (Chicago, IL, USA; cat. no. 10494-1-AP), and Boster Biological Technology, Ltd. (Wuhan, China; cat. no. BM1243), respectively. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, Alexa Fluor 555 anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA; cat. nos. 7074, 4413 and 4408, respectively). TRIzol Reagent and all of the cell culture media/reagents and salt solutions were obtained from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A SYBR PrimeScript™ RT-PCR kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China). Amersham ECL reagents for western blotting were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA).
4
Quantifying K-Ras Expression in Cell Lines
5
Antibody Panel for Cellular Signaling
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Primary antibodies for Phospho-S6 Ribosomal Protein ser235/236, mTOR, phospho-4EBP1, and GAPDH were from Cell Signaling, Phospho-FAK (Tyr397), HPDL from Thermo Fisher, Paxilin and LAMP2 from BD-bioscience; PAK1 from Proteintech. Secondary antibodies Alexa-fluor 594 anti-Rabbit IgG, Alexa-fluor 488 anti-Rabbit IgG, Alexa-fluor 594 anti-Mouse IgG and Alexa-fluor 488 anti-Mouse IgG were from Cell Signaling, IRDye 800CW and IRDye 680CW were from LI-COR. Alexa fluor TM 555 Phalloidin, Click-iT EdU Imaging Kits, CellEvent Caspase-3/7 Green Detection kit, NHS-Fluorescein, NHS-Alexa Fluor 555, pH-rodo iFL STP ester red and Hoechst 33342 were from Invitrogen. DRAQ5 was from LI-COR. Collagen I and Matrigel were from Corning. Geltrex was from Thermo Fisher. All media and dialyzed FBS were from Gibco, except for DMEM with no amino acid which was from US Biological life science and Plasmax which was kindly provided by Dr Tardito, CRUK Scotland Institute, Glasgow. The details of Plasmax composition were previously described by Vande Voorde and colleagues [18 (link)]. E64d (Aloxistatin) and PF573228 were from AdooQ Bioscience. GM6001 was from APEXBIO. Dynasore, Filipin, and EIPA were from Sigma. FRAX 597 was from bio-Techne. Nitisinone was from MCE. PP2 was from Generon. C13-tyrosine was from Sigma-Aldrich.
6
Visualizing CAR and YAP in CITCO-Treated HepG2 Cells
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According to previous publications24 (link), HepG2 cells (ATCC, Cat# HB-8065, RRID: CVCL_0027) were cultured in DMEM containing 10% FBS and then treated with DMSO or 10 μmol/L CITCO (Sigma–Aldrich, Cat# C6240) for 48 h. After incubation, cells were fixed in 4% paraformaldehyde and 0.5% Triton X-100, respectively. Then, cells were incubated with rabbit polyclonal anti-CAR (Abcam, Cat# ab62590, RRID: AB_956175 ) and mouse monoclonal anti-YAP (R&D Systems, Cat# MAB8094, RRID: AB_2819224 , MN, USA) overnight at 4 °C. Cells were stained with secondary antibodies including anti-mouse IgG Alexa Fluor 488 (Cell Signaling Technology, Cat# 4408, RRID: AB_10694704 ) and anti-rabbit IgG Alexa Fluor 647 (Cell Signaling Technology, Cat# 4414, RRID: AB_10693544 ). Images were acquired using a confocal microscope (Olympus FV3000, Tokyo, Japan).
7
Immunofluorescence Assay for PRRSV Detection
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PAMs or Marc-145 cells that were grown in 12-well cell plates or in 15 mm covered, glass-bottomed petri dishes were washed with PBS and were fixed with 4% paraformaldehyde (Beyotime, P0099) for 10 min, and they were then permeabilized with 0.5% Triton X-100 (Beyotime, P0096) in PBS for 5 min. The cells were blocked in Immunol staining blocking buffer (Beyotime, P0260) for 30 min at room temperature. Subsequently, the primary antibodies were incubated overnight at 4°C. After washing to remove unbound antibodies, the cells were incubated with the appropriate Alexa Fluor 488 or 555 conjugated secondary antibodies at room temperature for 1 h in the dark. Washing three times again, the cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI; Solarbio, C0065) for 5 min. Finally, the fluorescence images were taken using an inverted fluorescence microscope (NIKON ECLIPSE Ti2-U) or a confocal laser scanning microscope (TCS-SP5, LEICA). Antibodies used in the immunofluorescence and confocal microscopy: anti-Myc antibody (1:800; Cell Signaling Technology, 2276s), anti-PRRSV N antibody (4A5) (1:1500; Jeno Biotech, 9041), anti-clathrin heavy chain (1:1000, Abcam, ab21679), anti-Mouse IgG Alexa Fluor 488 (1:1000, Cell Signaling Technology, 4408s), and anti-Rabbit IgG Alexa Fluor 555 (1:1000, Cell Signaling Technology, 4413s).
8
HNP1, HNP2, and HNP3 in TGF-β-Mediated Cellular Responses
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The following reagents were used in this study: HNP1, HNP2 and HNP3 (Peptide Institute, Inc., Japan); TGF-β (BioLegend Inc., CA, USA); Dulbecco’s Modified Eagle’s Medium (Cytiva, Marlborough, MA, USA); Fetal Bovine Serum (Gibco, Grand Island, NY); ProLong™ Gold Antifade Mountant with DAPI (Invitrogen, CA, USA); LDH-Cytotoxicity Colorimetric Assay Kit II (BioVision Inc., CA, USA); RNeasy Mini Kit (QIAGEN Inc., Hilden, Germany); iScript Reverse Transcription Supermix, SsoAdvanced™ Universal Probes Supermix (Bio-Rad Inc., CA, USA); Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc., NY, USA); 1X Protease/Phosphatase Inhibitor Cocktail, Rabbit anti-COL1A1 antibody, Mouse anti-Ki-67 antibody, Rabbit anti-β-actin antibody, Mouse anti-rabbit IgG antibody (HRP conjugate), Anti-rabbit IgG Alexa Fluor 555, Anti-mouse IgG Alexa Fluor 488 (Cell Signaling Technology Inc., MA, USA); Amersham ECL Western Blotting Detection Kit (GE Healthcare Life Sciences Inc., MA, USA); Alliance Q9 chemiluminescence imaging system (Uvitec Inc., UK); Tissue-Tek O.C.T.™ Compound (Sakura, Alphenaan den Rijn, Netherlands).
9
Immunofluorescence analysis of transfected cells
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0:5×105 cells were seeded on poly-Lysine coated high-precision glass coverslips (18 mm round, #1.5) in 12-well culture plates one day prior to transfection. Transfection was performed as described for flow cytometry experiments. A total amount of 200 ng DNA (20 ng synTFs and 180 ng ssDNA) was used for transfection experiments. PEI was scaled to 12-well plate volume of 100 μL total transfection mix. 48 h post transfection, cells were washed with 1x PBS, fixed with 2% PFA (Fisher Scientific) and blocked for 30 min with 10% Goat serum (VWR) in 1x PBS after washing. Immunodetection was performed using anti-HA-tag (6E2) mouse monoclonal antibody (Cell Signaling) and anti-GFP (D5.1) rabbit monoclonal antibody (Cell Signaling) 1:200 in 1%BSA/PBS overnight. Cells were washed with 0.1% Triton X-100 and incubated with anti-mouse IgG Alexa Fluor 488 (#4408, Cell Signaling) and anti-rabbit IgG Alexa Fluor 647 antibodies 1:1000 in 1%BSA/PBS for 1 h. After washing with 0.1% Triton X-100, nuclei were stained with 2 μg/mL Hoechst-33342 (Thermo Fisher Scientific) and mounted on glass slides using Prolong Gold Antifade (Thermo Fisher Scientific). Image acquisition was performed at least 16 h after mounting coverslips on glass slides.
10
Immunofluorescence analysis of transfected cells
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