Cardiomyocytes were incubated with indicated doses of drugs. Next, cells were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.3% Triton X-100 for 1 h at room temperature. Immunofluorescence assessment of cardiomyocyte expression of GRP78 was carried out using cardiomyocyte-specific mouse monoclonal anti-α-actinin (1 : 100 in antibody dilution) and rabbit polyclonal anti-GRP78 (1 : 100 in antibody dilution), followed by staining with goat anti-mouse secondary Flour-594 antibody (1 : 200 in antibody dilution; Invitrogen, Carlsbad, CA, USA) and goat anti-rabbit secondary Alexa Flour 488 (1 : 200 in antibody dilution; Invitrogen). Cardiomyocyte expression of CHOP was carried out using mouse monoclonal anti-α-actinin (1 : 100 in antibody dilution) and rabbit polyclonal anti-CHOP (1 : 100 in antibody dilution), followed by staining with goat anti-mouse secondary Flour-594 antibody (1 : 200 in antibody dilution; Invitrogen) and goat anti-rabbit secondary Alexa Flour 488 (1 : 200 in antibody dilution; Invitrogen). The sections were observed and images were captured by confocal laser scanning microscopy (Nikon, Tokyo, Japan).
Alexa flour 488
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany, United Kingdom
Alexa Fluor 488 is a fluorescent dye commonly used in biological research applications. It is a green-fluorescent dye with excitation and emission maxima at 495 nm and 519 nm, respectively. Alexa Fluor 488 is known for its high brightness and photostability, making it a popular choice for various labeling and imaging techniques.
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Lab products found in correlation
Alexa flour 594, by Thermo Fisher Scientific (26 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (22 mentions)
Alexa flour 568, by Thermo Fisher Scientific (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (16 mentions)
Hoechst 33342, by Thermo Fisher Scientific (8 mentions)
Alexa flour 555, by Thermo Fisher Scientific (7 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (7 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (6 mentions)
Bovine serum albumin (bsa), by Merck Group (5 mentions)
Eclipse ti u, by Nikon (5 mentions)
Lsm 700, by Zeiss (5 mentions)
Prolong gold antifade, by Thermo Fisher Scientific (5 mentions)
Alexa fluor 647, by Thermo Fisher Scientific (4 mentions)
Vectashield mounting medium, by Vector Laboratories (4 mentions)
Triton x 100, by Merck Group (4 mentions)
Axio imager z1 microscope, by Zeiss (4 mentions)
Slowfade, by Thermo Fisher Scientific (4 mentions)
Lysotracker red, by Thermo Fisher Scientific (3 mentions)
Cyanine3 (cy3), by Jackson ImmunoResearch (3 mentions)
Lsm 780, by Zeiss (3 mentions)
232 protocols using alexa flour 488
1
Cardiomyocyte Expression of GRP78 and CHOP
2
Immunocytochemical Analysis of Stem Cell Markers
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Medium was aspirated and the films were rinsed in phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 20 minutes at 4 °C. Then the cells were permeabilized with 0.1% Triton X-100 for 10 min on ice. After rehydration in PBS and air drying, the films were blocked with 3% goat serum (Abcam) in PBS for 2 hours. Then primary antibody for cytokeratin 3 (1:100 in 3% goat serum; Millipore, #CBL218-I) was added to the membrane and kept in a humidified chamber overnight at 4 °C. Films were washed thrice for 15 minutes in PBS-T (Phosphate-Buffered Saline/Tween-20); Alexa Flour 488 (Invitrogen)-conjugated secondary antibody (1:500 in 3% goat serum) was added to each membrane and incubated for 60 minutes at 37 °C. After washing, films were counterstained with DAPI nuclear stain and mounted using mounting medium for fluorescent microscopy (DAKO), and sealed under a cover slip. The cells were viewed under a fluorescence microscope (ECLIPSE TE 2000, Nikon, Japan). Immunocytochemistry was also performed using ABCG2 (1:100 Sigma, AV 43649), vimentin (1:100, Santa Cruz Biotechnology, Sc-6260) primary antibody and Alexa Flour 488 (Invitrogen)-conjugated secondary antibody (1:500 in 3% goat serum) and viewed under a fluorescence microscope (DMI 4000B; Leica Microsystems).
3
Immunofluorescence Analysis of Psoriatic Skin
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Punch biopsies from NL and L plaque-type psoriatic skin were obtained as described before.1 (link),2 (link) Then, 4-µm sections were heated in 10 mM sodium citrate buffer for 10 minutes in a microwave oven (750 W). The sections were further permeabilized in Tris-buffered saline (TBS) with 1% Triton X-100 for 10 minutes and washed with TBS with 0.3% Triton X-100. Nonspecific binding sites were blocked with Image-iT FX signal enhancer (136933; Thermo Fisher Scientific, Waltham, MA, USA). Tissue sections were incubated with the primary antibody dissolved in TBS med 0.3% Triton X-100 (T-9284; Sigma-Aldrich) with 5% of goat nonimmune serum (DAKO X0907) at 4°C overnight. Antibodies were rabbit anti-P-RSK1 (T573) or P-RSK1 (S380; Cell Signalling Technology, Beverly, MA, USA), isotype control (sc-2027) normal rabbit IgG, and goat anti-rabbit Alexa Flour 594 (A-11037) or Alexa Flour 488 (A 31628; Invitrogen, Molecular Probes®). The samples were mounted in ProLong anti-fade reagent with 4′,6-diamidine-2′-phenylindole (DAPI; P36935; Invitrogen, Roskilde, Denmark). Samples were viewed by using an epifluorescence microscope (Leica Microsystems, Wetzlar, Germany).
In other experiments, tissue sections were incubated with rabbit anti-ILKAP (ab70019; Abcam, Cambridge, UK) at 4°C overnight, developed with goat anti-rabbit Alexa Flour 488 (A-31628; Invitrogen) for 1 hour, and mounted with DAPI.
In other experiments, tissue sections were incubated with rabbit anti-ILKAP (ab70019; Abcam, Cambridge, UK) at 4°C overnight, developed with goat anti-rabbit Alexa Flour 488 (A-31628; Invitrogen) for 1 hour, and mounted with DAPI.
4
Antibody Characterization for Signaling Pathways
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The following antibodies were used: anti-S100b (1:500, ref Z0311, Dako), anti-GFAP (1:500, ref M0761, Dako), anti-phosphocMet (Y1234/1235) (1:250, ref 3077, Cell Signaling), anti-phospho ERK1/2 (1:1000, ref 9106, Cell Signaling), anti-ERK(1:1000, ref 4695, Cell Signaling), anti-phospho S6K (1:500, ref 9205, Cell Signaling), anti-S6K (1:1000, ref 2708, Cell Signaling), anti-phospho AKT (1:1000, ref 13038, Cell Signaling), anti-AKT (1:1000, ref 9272, Cell Signaling), anti-phosphomTOR(1:1000, ref 2971, Cell Signaling), and anti-mTOR(1:1000, ref 2983, Cell Signaling), anti-HGF (1:500, ref MA5-14160, Thermo scientific), anti-GAPDH (1:5000, ref ab9485, Abcam), anti-cMet (1:1000, ref SAB4300599, Sigma), anti-Rabbit IgG secondary antibody Alexa Flour 555 (1:500, ref A-31572, Thermo Scientific), anti-Rabbit IgG secondary antibody Alexa Flour 488 (1:500, ref A-21206, Thermo Scientific), anti-mouse IgG secondary antibody Alexa Flour 555 (1:500, ref A-31570, Thermo Scientific), anti-mouse IgG secondary antibody Alexa Flour 488 (1:500, ref A-11006, Thermo Scientific), anti-mouse IgG HRP (1:10000, ref A0168, Sigma), and anti-rabbit IgG HRP (1:10000, ref RABHRP1, Sigma).
5
Subcellular Localization of Mitochondrial Dynamics Proteins
6
PI3Kγ Regulation of Phospho-ERK Imaging
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MEFs were transfected with vector or HA-tagged PI3Kγ and plated onto coverslips treated with poly l -lysine. Cells were serum-starved for 4 h, stimulated, fixed (4% para-formaldehyde), permeabilized with ice cold methanol for 10 min at –20°C, and blocked with 5% normal goat serum (NGS) in PBS. Anti-phospho ERK (1:1000; Cell Signaling) and/or anti-HA (1:100; Roche) were used as primary antibodies, while goat anti-rabbit AlexaFlour 488 (1:200; Molecular Probes) and anti-mouse AlexaFlour 568 (1:200; Molecular Probes) were used as secondary antibodies. Samples were visualized using sequential line excitation at 488 and 568 nm for green and red, respectively. Quantitation of the fluorescence intensity of the cells was performed using the IMAGE PRO PLUS 7 program (Media Cybernetics).
7
Immunofluorescent Labeling of Flavivirus Antigens
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The cultured cells were fixed in 4% paraformaldehyde for 20 min at room temperature and then incubated with methanol for 20 min at −20 °C. Fixed cells were blocked with 3% bovine serum albumin in PBS and then incubated with primary antibodies to Flavivirus Group Antigen (clone D1-4G2-4-15, Millipore, Billerica, MA), GFAP (Dako), or cleavage caspase 3 (Cell signaling Technology) overnight. At the second day, the cells were washed with PBS for three times and incubated for 1 h at room temperature with the secondary anti-mouse IgG antibody (coupled with green dye, Alexa Flour 488, Molecular Probes, Eugene, Oregon). Nuclear DNA were labeled with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, St. Louis, MO) for 10 min after the secondary antibody at room temperature. Cover slips were mounted on glass slides with mounting medium (Sigma-Aldrich). Fluorescent images were obtained using a Zeiss 710 Confocal Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany). All obtained images were imported into Image-ProPlus, version 7.0 (Media Cybernetics, Sliver Spring, MD) to quantify the number of infected cells. The assessors were blinded during image acquisition or quantification.
8
Antibody Generation and Characterization
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A rabbit polyclonal antibody for hCRB1 was raised against a GST fusion protein corresponding to amino acids 255–407 within the extracellular domain of the protein. hMIB1 and mEPB41L5 polyclonal antibodies were raised against GST fusion proteins corresponding to amino acids 236–445 and 669–732 of the proteins respectively, and antisera affinity purified using the corresponding antigen. Additional primary antibodies used available from commercial sources include: HA12CA5 (Roche,IF 1:500, IB 1:1000), HA-Tag (C29F4; Cell Signaling Technology 3724; IP 5μl), Histidine (HIS.H8, Sigma-Aldrich 05-949, IB 1:500), FLAGM2 (Sigma-Aldrich, mouse, IF 1:500, IB 1:1000; Abnova, rabbit, IF: 1:500), myc9E10 (Clontech,; IF 1:500, IB 1:1000), T7tag (EMD Millipore 69522-3; IB 1:500) E-cadherin (BD Biosciences, mouse, IF 1:300), ZO1 (Zymed, mouse, IF 1:300; Millipore, rat, IF 1:500), β-catenin (Millipore, rabbit, IF 1:300), Actin-phalloidin (Molecular Probes, Invitrogen, IF 1:500), GFP (ThermoFisher Scientific A-11122; IF 1:500) and CRB3 (Sigma HPA013835, IF 1:50). Secondary antibodies conjugated with AlexaFlour 488 (Molecular Probes), Cy3 and Cy5 (Jackson Laboratories).
9
Immunofluorescence Staining Protocol
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For IF experiments, cells were transfected and, after 24 h, were replated on coverslips and then incubated for another 24 h. Cells were fixed in 2% paraformaldehyde for 10 min at room temperature and permeabilized for 10 min with 0.1% sodium azide phosphate-buffered saline (PBS), 0.1% Triton-X, and 5% bovine serum albumin (BSA). Primary and secondary antibodies were diluted in 0.1% sodium azide PBS, 0.2% Triton-X, and 0.5-1% BSA. As secondary antibodies, anti-rabbit Alexa Flour 488 and anti-mouse Alexa Flour 568, from Molecular Probes, were used. Cells were counterstained with DAPI (4',6-diamidino-2-phenylindole; Sigma) and slides were mounted on Vectashield (Vector Laboratories). Labeled cells were imaged using a Zeiss LSM510 Meta Confocal Laser Scanning Microscope.
10
LHRH Receptor Expression in Pancreatic Cancer Cells
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Example 6
One million cells from each of the pancreatic cancer cell lines were collected and washed with PBS containing 1% FBS. The cells were then incubated on ice for 30 min with 10 μg/ml of monoclonal mouse antibody against LHRH receptor. The cells were washed twice with PBS containing 1% FBS, and then were further incubated for an additional 30 min on ice with Alexa Flour 488 (Molecular probes, Eugene, Oreg.), a goat anti-mouse secondary antibody conjugated to FITC. The cells were washed twice more, and suspended in 300 μl of PBS for flow cytometry. 10,000 events were collected on a FASCcalibur flow cytometer, and analyzed using Cellquest software (Becton Dickinson). Isotype controls were prepared similarly, except that an IgG1 antibody from Santa Cruz Biotechnology (Santa Cruz, Calif.) was used instead of an LHRH receptor antibody. Results showed that the LHRHR-mab specifically bound to the pancreatic cancer cell surfaces (data not shown).
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