YPD medium was used for growth of Saccharomyces cerevisiae strain EBY100, SD-CAA, for selection of pYD2-transformed EBY100 and SG-CAA, and for induction of scFv expression on the surface of EBY100. Escherichia coli strain DH5α was used for subcloning and preparation of plasmid DNA. Pure holotoxin BoNT/E3 was purchased from Metabiologics (Madison, WI, USA). Crude extract complex for subserotypes E2 and E4 was obtained from the U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID). Pure complex subserotype E1 was purchased (Wako chemicals USA Inc., Richmond, VA, USA). All IgGs were expressed from Chinese hamster ovary (CHO) cells, while the mouse anti-SV5 antibody was purified from hybridoma cells and labeled with an AlexaFluo-488 or AlexaFluo-647 labeling kit (Invitrogen, Carlsbad, CA, USA). The secondary antibody, PE-conjugated goat anti human-Fc, F(ab) was purchased (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).
Alexa fluo488
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 is a fluorescent dye commonly used in biomedical research applications. It is designed to emit green fluorescence when excited with a 488 nm light source. The dye can be used to label a variety of biomolecules, including proteins, nucleic acids, and small molecules, for detection and visualization purposes.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (3 mentions)
Alexafluo 647 labeling kit, by Thermo Fisher Scientific (2 mentions)
Fluorescence microscope, by Olympus (2 mentions)
Av300 asw confocal microscope, by Olympus (1 mentions)
Alexa fluor 488 donkey anti goat igg, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd streptavidin, by LI COR (1 mentions)
Alexa fluor 568 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Goat anti mouse igg2b alexa fluor 647, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 568 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor568 goat anti mouse igg2a, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti mouse igg2a, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 goat anti mouse igg1, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 647 donkey anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Goat anti human fab, by Jackson ImmunoResearch (1 mentions)
Goat anti chicken igy, by Abcam (1 mentions)
Cm3050, by Leica (1 mentions)
14 protocols using alexa fluo488
1
Yeast Surface Display of BoNT/E Antibodies
2
Immunofluorescent Visualization of LC3 Puncta
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Cells were cultured on cover glasses in a 6-well plate and then washed with PBS 3 times. The cells were fixed in 4% paraformaldehyde for 30 min at room temperature and permeabilized with 0.5% Triton X-100 in PBS for 20 min. Then, the cells were blocked with 5% BSA for 60 min at room temperature and incubated with rabbit anti-LC3A/B antibody at 4°C overnight. On the second day, washed the cells twice with PBS and incubated them with anti-rabbit Alexa Fluo488 (Invitrogen) for 60 min at a room temperature in the dark. Finally, the cells were mounted on the cover glasses with 4',6-diamidino-2-phenylindole (DAPI) (Invitrogen), and the image was obtained on a fluorescence microscope (Olympus Inc., USA). The number of LC3 puncta was qualified manually.
3
Liraglutide's Effect on Adipocyte Mitochondria
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Adipocyte-inducing 3T3-L1 cells were treated with liraglutide for 3 days as described above. Cells were fixed in 4% paraformaldehyde and permeabilized by 1% TritonX-100. Following blocking with 1% bovine serum albumin, cells were serially incubated in rabbit anti-COX-IV and Goat anti-rabbit Alexa Fluo488 (Invitrogen Corporation, Carlsbad, CA, USA). The images were acquired using the AV300-ASW confocal microscope (Olympus America Inc., Center Valley, USA). Image magnification: 100×.
4
Caspase-3 and H2AX Activation Assays
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Analysis of caspase-3 activation was performed using Caspase-3 fluorescent assay kit (Cayman chemical). For quantification of cell death, cells were re-suspended in 500 μL of PBS and stained with propidium iodide (PI). Fluorescence was determined with a FACScan™ (Beckman Dickinson). To detect H2AX phosphorylation cells were fixed in paraformaldehyde 3% for 20′ at room temperature, permeabilized with 0.1% TritonX-100 and incubated with the primary antibody anti-pH2AX for 1hr. After washes, cells were incubated with the secondary antibody anti-rabbit Alexa Fluo-488 (Invitrogen) for 30 min. Results were from al least 3 experiments ± SD.
5
Multicolor Immunofluorescence Labeling Protocol
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We use the following secondary antibodies, and their conditions of use include: Alexa Fluor 488 Goat anti-Rabbit IgG (Invitrogen, A-11008, 1:500); Alexa Fluor 647 Goat anti-Mouse IgG1 (Invitrogen, A-21240, 1:500); Alexa Fluor 647 Goat anti-Mouse IgG2b (Invitrogen, A-21242, 1:500); Alexa Fluor 568 Goat anti-Mouse IgG2a (Invitrogen, A-21134, 1:500); Alexa Fluor 647 Goat anti-Rabbit IgG (Invitrogen, A27040, 1:500); Alexa Fluor 647 Donkey anti-Rabbit IgG (Invitrogen, A-31573, 1:500); Alexa Fluor 568 Donkey anti-Rabbit IgG (Invitrogen, A10042, 1:500); Alexa Fluor 647 Donkey anti-Mouse IgG (Invitrogen, A-31571, 1:500); Alexa Fluor 568 Donkey anti-Mouse IgG (Invitrogen, A10037, 1:500); Alexa Fluor 488 Donkey anti-Goat IgG (Invitrogen, A-11055, 1:500), Alexa Fluor 488 Goat anti-Mouse IgG2a (Invitrogen, A-21131, 1:500), Alexa Fluor 488 Goat anti-Rat IgG (Invitrogen, A-11006, 1:500); Streptavidin, Alexa Fluo 488 (IF: 1:500, S11223, Invitrogen); IRDye 680RD Streptavidin (WB: 1:3000, 926-68079, LiCor).
6
Yeast Surface Display of scFv Antibodies
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YPD medium was used for growth of Saccharomyces cerevisiae strain EBY100, selective growth dextrose casamino acids media (SD-CAA) for selection of pYD4 transformed EBY100 and selective growth galactose CAA media (SG-CAA), for induction of scFv expression on the surface of EBY100. E. coli DH5α was used for subcloning and preparation of plasmid DNA, BL21, for BoNT fragments and GST-fused SNAP25 (141–206) expression and E. coli TG1 cells, for scFv-displaying phage packaging and soluble scFv expression. Pure holotoxins BoNT/A1 and A2 were purchased from Metabiologics (Madison, WI). All BoNT IgGs were expressed in Chinese hamster ovary cell (CHO) cells, while the mouse anti-SV5 antibody was purified from hybridoma and labeled with AlexaFluo-488 or AlexaFluo-647 labeling kit (Invitrogen, Carlsbad, CA). All the secondary antibodies including PE or APC-conjugated goat anti human-Fc, goat anti-mouse Fc and goat anti-human Fab were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
7
Immunohistochemical Analysis of Bone Marrow
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After fixation, samples were decalcified in 20% EDTA at 4 C for 1 week. Samples were embedded in OCT for frozen sectioning. 10mm sections were obtained. Sections were incubated with primary antibody at 4 C O/N. The following primary antibodies were used in this study: Anti-CD44, Anti-CD73, Anti-CD146, Anti-Periostin, Anti-Col I, Anti-Sp7, Anti-ß3-tublin, Anti-aSMA, Anti-LepR, Anti-ß-gal, Anti-SOST, Anti-Perilipin-1, Anti-CD31, Anti-CD34, and Anti-Nestin, Anti-NG2, Anti-PDGFRa, Anti-SHH. The secondary antibodies included goat anti-mouse (Invitrogen A11029, 1:100), anti-rat (Invitrogen A11006), anti-rabbit (Invitrogen A11034) IgG conjugated to Alexa 488, and goat anti-chicken IgY conjugated to AlexaFluo 488 (Abcam ab150169).
GS-IB4 (Isolectin GS-IB4 from Griffonia Simplicifolia) has been widely used for labeling endothelial cells in various organs including long bone marrow, lung, heart and retina (Epah et al., 2018; Hooper et al., 2009; Xu et al., 2018; Ohle et al., 2012; Arnold et al., 2012) . For periodontium vasculature staining, mouse samples were fixed with 4% PFA overnight and decalcified in 20% EDTA for one week. Samples were embedded in OCT and 20mm sections were obtained. Sections were incubated with GS-IB4 conjugated with Alexa-Fluo488 (Invitrogen) at 1:100 dilution at 4 CC O/N. Sections were then briefly washed with PBS and counterstained with DAPI for imaging.
GS-IB4 (Isolectin GS-IB4 from Griffonia Simplicifolia) has been widely used for labeling endothelial cells in various organs including long bone marrow, lung, heart and retina (Epah et al., 2018; Hooper et al., 2009; Xu et al., 2018; Ohle et al., 2012; Arnold et al., 2012) . For periodontium vasculature staining, mouse samples were fixed with 4% PFA overnight and decalcified in 20% EDTA for one week. Samples were embedded in OCT and 20mm sections were obtained. Sections were incubated with GS-IB4 conjugated with Alexa-Fluo488 (Invitrogen) at 1:100 dilution at 4 CC O/N. Sections were then briefly washed with PBS and counterstained with DAPI for imaging.
8
Multimodal Labeling of Neuronal Subpopulations
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Mice underwent deep anaesthesia using isoflurane and were subsequently intracardially with PBS (phosphate buffered saline) followed by 4% paraformaldehyde (PFA). Brain samples were fixed in 4% PFA at 4 °C overnight and then dehydrated in a PBS solution containing 30% sucrose at 4 °C until tissues sank. Brain slices, with a thickness of 40 μm (Leica CM3050S), were prepared and stored in cryoprotectant solution at 4 °C. Following three PBS washes, slices were blocked with 1% BSA for 1 h at room temperature. The primary antibodies (Rabbit-anti-GFP, 1:1000, Thermo Fisher Scientific, A11122; Rabbit-anti-NPY, 1:1000, Cell Signalling Technology, 11976S; Mouse-anti-GAD1, 1:1000, Millipore, MAB5406) were incubated at 4 °C overnight, followed by three PBS washes. Subsequently, slices were exposed to the secondary antibodies (Alexa Fluo 488, 1:1000, Invitrogen, A11034; Alexa Fluo 568, 1:1000, Invitrogen, A11031) at room temperature for 1 h. After three PBS washes, the slices were mounted for imaging using an Andor spinning disk confocal microscope. Image processing and analysis were conducted using ImageJ and Imaris software.
9
Immunofluorescence Imaging of PrPSc in Cells
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Immunofluorescence staining of prion-infected cells was adapted from previously reported protocols (56 (link), 57 ) with some modifications. Cells were grown on poly-d -lysine-coated coverslips in a 24-well plate. At approximately 50% confluence, cells on coverslips were gently washed twice with 1× PBS, fixed in 4% paraformaldehyde at room temperature for 30 min, and then permeabilized with 0.5% Triton X-100 for 5 min. To detect PrPSc, cells were digested with 20 μg/ml of PK at 37 °C for 10 min and incubated with 2 mM of PMSF for 15 min to terminate the reaction, and then incubated with 6 M guanidine hydrochloride at room temperature for 15 min. After that, cells were blocked with 5% normal goat serum in 1× PBS and then reacted sequentially with 3F10 anti-PrP antibody and then goat antimouse IgG Alexa Fluo-488 (Invitrogen; catalog number: A10680). Nuclei were stained with 1 μg/ml 4′,6-diamidino-2-phenylindole (Invitrogen; catalog number: D3571). Confocal laser scanning microscopy (Fluoview FV10i; Olympus) was performed. The fluorescence intensity (Fi) of stained PrP proteins was calculated as total Fi/cell numbers using ImageJ (National Institutes of Health). Relative PrP convertibility was calculated as Fi(+PK)/Fi(−PK).
10
Immunofluorescence Assay of DNA Damage and Autophagy
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Cells were seeded in 24-well plates and cultured for 24 h, washed twice with PBS, fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 (Sigma-ALDRICH, America), and 3% bovine serum albumin was used to block nonspecific antigen-antibody reaction. After incubation of primary anti-γH2AX antibody or anti-LC3 antibody overnight and washing three times with PBST, cells were incubated with donkey anti-rabbit Alexa Fluo488 (Invitrogen, America) for 1 h at room temperature. Cells were rinsed and stained with DAPI (Invitrogen, America). The immunofluorescence signal was detected by a fluorescence microscope (Olympus Inc., America).
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