Mouse thoracic aortas were isolated and frozen sections were blocked in 5% goat serum 37 °C for 1 h, and then incubated at 4 °C overnight with the antibody against α-SMA (Cat. No. 14395; 1:100 dilution, Proteintech), FAM3B (Cat. No. sc-83250; 1:50 dilution, Santa Cruz) or SM-MHC (Cat. No. sc-6956; 1:50 dilution). To detect the co-localization of SM-MHC and FAM3B (Fig. 1B ), tissue sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 (Cat. No. 4408; 1:500 dilution, Cell Signaling Technology) and Alexa Fluor 555 (Cat. No. 4413; 1:500 dilution, Cell Signaling Technology) for 30 min at room temperature. For Fig. S1A , secondary antibodies conjugated to Alexa Fluor 488 (Cat. No. 4412; 1:500 dilution, Cell Signaling Technology) and Alexa Fluor 555 (Cat. No. 4413; 1:500 dilution, Cell Signaling Technology) were instead used. Nuclei were identified with DAPI. Non-immune IgG was used as a negative control. The sections were photographed with a DP70 digital camera connected to a microscope (Olympus BX41).
Alexa fluor 555
Manufactured by Cell Signaling Technology
Sourced in United States
Alexa Fluor 555 is a fluorescent dye used in various applications, such as immunofluorescence, flow cytometry, and fluorescence microscopy. It has an excitation maximum at 555 nm and an emission maximum at 565 nm, making it suitable for detection with standard fluorescence filter sets. Alexa Fluor 555 is a bright and photostable dye that can be conjugated to a wide range of biomolecules, providing a valuable tool for the visualization and analysis of biological samples.
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Lab products found in correlation
Alexa fluor 488, by Cell Signaling Technology (16 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (4 mentions)
Inverted microscope, by Zeiss (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Hrp labeled secondary antibody, by Cell Signaling Technology (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Abcam (2 mentions)
Ab15580, by Abcam (2 mentions)
Eclipse te2000 u, by Nikon (2 mentions)
Foxa2, by Abcam (2 mentions)
Fluoro gel aqueous mounting media, by Electron Microscopy Sciences (2 mentions)
Alexa fluor 568, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by BioLegend (2 mentions)
Epcam, by BD (2 mentions)
Lsm 780, by Zeiss (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Apotome, by Zeiss (2 mentions)
Antifade reagent, by Thermo Fisher Scientific (2 mentions)
Prolong gold antifade mountant with dapi, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Vectashield with dapi, by Vector Laboratories (1 mentions)
60 protocols using alexa fluor 555
1
Immunofluorescence Staining of Mouse Thoracic Aorta
2
Immunofluorescence Imaging of Myeloma Cells
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Myeloma cells were air-dried and fixed in 3.7% formaldehyde in phosphate-buffered saline (PBS) for 10 min at room temperature on glass slides using Shandon cytospin 2 (Thermo Fisher Scientific). Fixed cells were permeabilized, reduced and denatured for 30 min at room temperature in PBS buffer containing 0.5% SDS, 5% β-mercaptoethanol and 10% FBS. Then, cells were washed three times with PBS containing 4% FBS and 0.1% Triton X-100 (PET buffer) and incubated with the anti-FLAG antibody (1:200 dilution) and either rabbit anti-ILF2 (1:200 dilution), or anti-SAFB (1:200 dilution) antibody for 1 h. Cells were then washed three times with PET buffer and incubated with Alexa Fluor 555 and Alexa Fluor 647 labeled secondary antibodies (A11078; A11037 Cell Signaling Technology) for 1 h in the dark. All antibodies were diluted with 3% BSA and 0.5% Tween in PBS. Slides were mounted in VECTASHIELD with DAPI (Vector Labs) and observed with a confocal laser scanning microscope (BZ-X800, KEYENCE).
3
Automated Immunostaining with Confocal Imaging
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Automated immunostaining was carried out using Ventana BenchMarkXT platform (Ventana). The following antibodies were used; anti-vimentin and anti-cytokeratin (Ventana), rabbit anti-LC3A and rabbit anti-LC3B. Immunofluorescence was performed as described previously40 (link). For immunofluorescence, the following primary antibodies were used at the indicated concentrations: rabbit anti-LC3A (1:50) (Cat # 4599, Cell Signaling Technology), rabbit anti-LC3B (1:50) (Cat # 3868, Cell Signaling Technology), rabbit anti-vimentin (1:50) (Cat # 5741, Cell Signaling Technology), mouse anti-vimentin (1:100) (Cat # ab8978, Abcam), mouse anti-LAMP2 (1:50) (Cat # sc-18822, Santa Cruz Biotechnology), and mouse anti-LC3B (1:50) (Cat # sc-271625, Santa Cruz Biotechnology). Bound antibodies were visualized using Alexa Fluor 555 or Alexa Fluor 488 secondary antibodies (1:500) (Cell Signaling Technology). Cells were then counterstained using 4, 6-diamidinophenylindole (DAPI). Images were acquired using LSM 710 confocal scanning laser microscope (Carl Zeiss).
4
Comprehensive Immunostaining Protocol
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Anti-BRCA1 (#14823), anti-Ki67 (#9449), Alexa Fluor® 488 (#8878) and Alexa Fluor® 555 (#8953) antibodies were purchased from Cell Signaling Technology (Danvers, MA). Anti-Rictor antibody (A300-459A) was purchased from Bethyl Laboratories (Montgomery, USA). Anti-γH2AX (05-636) antibody and anti-Rad51 (PC-130) antibody were obtained from EMD Millipore (Norwood, USA). Anti-β-actin antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). pDR-GFP (#17617), pCBASceI (#26477) and Rictor shRNA plasmids (#1853 and #1854) were obtained from Addgene (Watertown, MA, USA). Olaparib was purchased from Selleckchem (Houston, TX).
5
Neuronal Markers and Signaling Pathways
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CaCl2 was purchased from Bodi Chemical Co., Ltd. (Tianjin, China). Antibodies specific against NeuN and Alexa Fluor-488, Alexa Fluor-555, and HRP-labeled secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States). S(+)-Ketamine (60 mg/kg, for 1 h), SC-51322 (30 nM, for 12 h), PF-04418948 (100 nM, for 12 h), and DG-041 (60 nM, for 12 h) were obtained from R&D Systems (Minneapolis, MN, United States), and CJ-42794 (40 nM, for 12 h) was obtained from MedChem Express (Monmouth Junction, NJ, United States). High-fidelity (HF) restriction enzymes for EcoRI, BamHI, XhoI, and AgeI were purchased from New England Biolabs (Beverly, MA, United States). DAPI was procured from Beyotime Institute of Biotechnology (Haimen, China). The plko.1-puro, psPAX2, pMD2.G, and plvx-IRES-zsgreen vectors were purchased from Addgene (Sidney, SD, United States). All the reagents used for the quantitative (q)RT-PCR and SDS-PAGE experiments were purchased from Bio-Rad Laboratories (Hercules, CA, United States), and all other reagents were obtained from Invitrogen (Carlsbad, CA, United States), unless otherwise specified.
6
Rofecoxib-Induced Neuroinflammatory Responses
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The COX-2 inhibitor rofecoxib was obtained from Sigma-Aldrich (St. Louis, MO, United States). Antibodies specific for β-actin (rabbit, 1:5000), COX-2 (rabbit, 1:3000), NeuN (mouse, 1:5000), glial fibrillary acidic protein (GFAP) (rabbit, 1: 5000), IL-1β (rabbit, 1:4000), TNF-α (rabbit, 1:4000), Alexa Fluor-488 (1:400), Alexa Fluor-555 (1:400), and HRP-labeled secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, United States). DAPI was obtained from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). An Iba1 antibody (rabbit, 1:4000) was purchased from Wako Life Sciences (Wako, Tokyo, Japan). All reagents for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) experiments were purchased from Bio-Rad Laboratories (Shanghai, China). All other reagents were from Invitrogen unless otherwise specified.
7
Immunofluorescence Localization of Phosphoryl-NF-κB
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Immunofluorescence was used to determine phosphoryl-NF-κB p65 localization. GH3 cells were fixed with paraformaldehyde (v/v, 1/25) at 37°C for 10 minutes. They were then permeabilized with cold acetone at −20°C for 3 minutes. After a PBS wash (0.1 mM, pH 7.4), cells were saturated with 3% BSA in PBS for 30 minutes, and incubated with the anti-phosphoryl-NF-κB p65 antibody (diluted 1 : 100) at 4°C overnight. After another PBS wash (0.1 mM, pH 7.4), the cells were incubated with the secondary antibody for 30 minutes at room temperature. Coverslips were washed twice with PBS (0.1 mM, pH 7.4), incubated with the goat anti-mouse IgG antibody conjugated with Alexa Fluor 555 (Cell Signaling Technology, Danvers, MA, USA) for 30 minutes in the dark, incubated in 5 μM DAPI staining solution (Invitrogen) for 5 minutes, and then washed in PBS. The fluorescence was monitored using an UltraVIEW VoX confocal system (PerkinElmer, Co., Norwalk, CT, USA).
8
Multiplexed Immunofluorescence Imaging of Lamellar Tissue
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Ten micrometer cryosections of lamellar tissue were fixed in 4% formaldehyde (Polysciences, Inc, Warrington, Pennsylvania) for 15 minutes at room temperature, cell membranes were permeabilized with ice‐cold methanol (Fisher Scientific, Waltham, Massachusetts) for 20 minutes at −20°C, and sections were blocked with 5% goat serum (Cell Signaling Technology, Inc.) in a phosphate‐buffered saline solution containing 3% Tween‐20 for 1 hour at room temperature. The slides were incubated overnight with a rabbit monoclonal antibody against P‐STAT3 (Y705) (used at a 1 : 100 dilution) and a mouse monoclonal antibody against total STAT3 (used at a 1 : 1500 dilution) (Cell Signaling Technology, Inc). After washing, the slides were incubated at room temperature for 1.5 hours in an anti‐rabbit monoclonal antibody conjugated with Alexa Fluor 555 (Cell Signaling Technology, Inc) (1 : 200 dilution) and an anti‐mouse monoclonal conjugated with Alexa Fluor 647 (1 : 500 dilution), then with the DNA‐intercalating dye 4′,6‐diamidino‐2‐phenylindole (Cell Signaling Technology, Inc); coverslips were applied, and the sections were digitally imaged using a laser assisted confocal microscope (Olympus, Center Valley, Pennsylvania).
9
Immunofluorescence Analysis of Transfected Cells
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IF analysis of the transfected cells (after 48 h) was performed as described previously (Yamasaki et al., 2016 (link)). The primary antibody was the abovementioned antibody, and the secondary antibodies were conjugated to goat anti-rabbit IgG (Alexa Fluor® 555, #4413S, Cell Signaling Technology, United States). Images were captured with a confocal laser microscope (LSM 780, Zeiss, Germany) with a 40 × objective.
10
Paraformaldehyde Perfusion and VCAM-1 Immunofluorescence
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Mice were perfused through the left ventricle with 4% (wt/vol) paraformaldehyde as described [26] (link). Briefly, the aortic root was embedded in OCT medium, frozen and cut in 10 μm-sections onto Fisher Superfrost Plus-coated slides, fixed and incubated with anti-VCAM-1 antibody (Southern Biotech; 1:100) followed by anti-rat Alexa-fluor 555 (Cell Signaling, 1:2000, 1 h). In the negative control, VCAM-1 antibody was replaced by an equivalent dilution of normal rat serum (NRS).
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