Dulbecco's modified Eagle's medium (DMEM), Roswell Park Memorial Institute (RPMI) medium, fetal bovine serum (FBS) and penicillin/streptomycin (P/S) were purchased from Welgene Inc. WST-1 and bovine serum albumin (BSA) were purchased from Sigma-Aldrich; Merck KGaA. A TUNEL assay kit was obtained from Promega Corporation. Hoechst dye, Mito-tracker™ Red CMXRos and the Alexa-Fluor 488-conjugated donkey anti-Rabbit antibody were purchased from Thermo Fisher Scientific, Inc. The anti-Ki-67 antibody was purchased from Abcam (cat. no. ab15580). The aspirin conjugated DK143 [(E)-2-(4-(2,3-dimethoxyphenyl)acryloyl)-4-methoxyphenyl-2-acetoxybenzoate, AS-DK143] and AS-DK143-loaded methoxy poly(ethylene glycol)-poly(L-lactide) NPs (AS-DK143-NPs) were prepared and characterized according to our previous report (22 (link)). HeLa cell line, HepG2, which is a hepatoblastoma cell line, and AGS cell line were purchased from the Korean Cell Line Bank; Korean Cell Line Research Foundation. BXPC-3 cells were obtained from the Japanese Collection of Research Bioresources Cell Bank.
Alexa fluor 488 conjugated donkey anti rabbit antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488-conjugated donkey-anti-rabbit antibody is a secondary antibody reagent designed for use in immunofluorescence applications. It is conjugated to the Alexa Fluor 488 dye, which has a green fluorescent emission. This antibody is specific for binding to rabbit primary antibodies.
Automatically generated - may contain errors
Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Tcs sp5 confocal microscope, by Leica camera (2 mentions)
Fluorescence mounting medium, by Agilent Technologies (2 mentions)
Rabbit anti trpc1 antibody, by GenScript (2 mentions)
Mouse anti plcβ1 sc 5291, by Santa Cruz Biotechnology (2 mentions)
Mouse anti β actin antibody a1978, by Merck Group (2 mentions)
Tunel assay kit, by Promega (1 mentions)
Hoechst dye, by Thermo Fisher Scientific (1 mentions)
Hela cell line, by Korean Cell Line Bank (1 mentions)
Ags cell line, by Korean Cell Line Bank (1 mentions)
Bxpc 3 cells, by Japanese Collection of Research Bioresources Cell Bank (1 mentions)
Anti ki67 antibody, by Abcam (1 mentions)
Mitotracker red cmxros, by Thermo Fisher Scientific (1 mentions)
Hepg2, by Korean Cell Line Bank (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Wst 1, by Merck Group (1 mentions)
Penicillin streptomycin, by Welgene (1 mentions)
Dulbecco s modified eagle s medium, by Welgene (1 mentions)
Fetal bovine serum (fbs), by Welgene (1 mentions)
Facscanto 2, by BD (1 mentions)
29 protocols using alexa fluor 488 conjugated donkey anti rabbit antibody
1
Aspirin-conjugated NPs for anti-cancer effects
2
SARS-CoV-2 RBD Antibody Staining
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dTK-S- or dTK-RBD-infected Vero cells were permeabilized with 0.1% Triton X-100 and incubated with the anti-RBD antibody for 1 h, followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit antibody (Thermo Fisher Scientific, Waltham, MA, USA; A11034). Samples were analyzed with FACS Canto II (BD Biosciences, San Jose, CA, USA).
3
Visualizing SARS-CoV-2 Spike Protein Expression
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HeLa cells were infected with VACV recombinants expressing SARS-CoV-2 spike protein or RBD for 24 h and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized with 0.2% Triton X-100 for 10 min. Permeabilized and un-permeabilized cells were blocked with 5% bovine serum albumin (BSA) and then incubated with the anti-RBD antibody for 1 h, followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit antibody (Thermo Fisher Scientific, A11034) for 1 h. Nuclei were stained by DAPI. Images were captured with an inverted confocal microscope Leica TCS SP5 (Leica Microsystems, Wetzlar, Germany).
4
Imaging Apoptosis in Drosophila Wing
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Wing imaginal disc staining and analysis was performed as described in [34 (link)], staining actin in red and activated caspase 3 in green. Nuclei were stained with ToPro3 (Thermo Fisher Scientific, Waltham, MA, USA) following instructions provided by the manufacturer, rabbit anti‐caspase 3 (Cell Signaling Technology, Danvers, MA, USA) was used at 1 : 50 dilution and detected with Alexa Fluor 488‐conjugated donkey anti‐rabbit antibody (Thermo Fisher Scientific, Waltham, MA, USA) at 1 : 2000 dilution. Actin was detected with Alexa Fluor 647‐conjugated phalloidin (Thermo Fisher Scientific, Waltham, MA, USA) at 1 : 50 dilution.
5
Quantification of Neuronal Differentiation in PC12 Cells
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At 1, 3, and 5 days after transfection, PC12 cells were washed with PBS for 5 minutes and fixed with 4% paraformaldehyde for 15 minutes. Cells were permeabilized with 0.3% Triton X-100 for 15 minutes and incubated with PBS containing 5% bovine serum albumin for 2 hours at room temperature. The fixed cells were washed 3 times with PBS containing 0.1% Triton X-100 and incubated overnight with anti-microtubule-associated protein 2 (MAP-2; Cat# AB5622, polyclonal antibody, rabbit anti-rat, 1:1000; Millipore, Bedford, MA, USA) at 4°C. The cells were incubated with an Alexa Fluor 488-conjugated donkey anti-rabbit antibody (Cat# A21206, 1:500, Thermo Fisher Scientific, Waltham, MA, USA; excitation and emission wavelengths of 488 and 525 nm, respectively) at room temperature for 2 hours. Nuclei were stained with DAPI (1:5000, Cell Signaling Technology; excitation and emission wavelengths of 340 and 488 nm, respectively) for 15 minutes. At least 6 microscopic fields from each sample were observed under a fluorescence microscope, and images were obtained at 40× (Leica LAS X software, Wetzlar, Germany). Five PC12 cells with the longest protrusion lengths were selected from at least 100 cells, the mean fluorescence density was calculated for each visual field, and the average protrusion length was measured by ImageJ software 1.8.0 version.
6
Immunofluorescence Visualization of NF-κB p65
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TNF-α-stimulated or unstimulated cell lines grown on chamber slides were fixed with 3.7% PBS-buffered formaldehyde and permeabilized with PBS/0.1% Triton-X100. Nonspecific binding was blocked with PBS/1% Aurion BSA-c (DAKO, Glostrup, Denmark). Coverslips were subsequently incubated for 1 hr with rabbit anti-NF-κB p65 (C22B4) mAb (Cell Signaling Technology, Frankfurt a.M., Germany) diluted 1:1,000 in PBS/1% Aurion BSA, followed by 3 x 5 min washes with PBS. Subsequently, the samples were incubated with Alexa Fluor 488 conjugated donkey anti-rabbit antibody (Invitrogen, Karlsruhe, Germany) diluted 1:500, in PBS/1% Aurion BSA-c. for 30 min. Samples were washed 2x for 5 min in PBS, stained with 300 nM DAPI (Sigma, Deisenhofen, Germany) for 1 min, followed by a brief wash with PBS and slide mounting, Slides were analyzed with a confocal microscope (Axioskop; Carl Zeiss, Göttingen, Germany) with × 40 magnification and a 0.75 numerical aperture oil immersion objective.
7
Immunostaining of Frozen Tumor Sections
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Three-micrometer frozen section of the treated mice were prepared, fixed and stained with rat-anti-mouse Gr1 antibody (BD PharMingen, 1:50), rabbit-anti-mouse cleaved-Caspase-3 antibody and rabbit-anti-mouse A20 antibody (Abcam, 1:400). The secondary antibodies were Alexa Fluor 488-conjugated donkey-anti-rabbit antibody (Invitrogen, 1:500) and Alexa Fluor 594-conjugated donkey-anti-rat antibody (Invitrogen, 1:100). Samples were also stained with DAPI (5 μg/ml) for 10 minutes, washed and were visualized by a Leica TCS SP5 confocal microscope. Negative control: tumor sections were stained with PBS and secondary antibodies without primary antibodies.
8
Immunofluorescence Characterization of PPAR Expression in Brain
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Immunofluorescence to characterize PPAR expression in brain sections was performed using sequential day staining. After 3 × 10 min. washes in PBS, nonspecific binding was blocked by incubating for 1 h at room temperature in a blocking buffer containing dilution media, 5% normal serum, and 0.5% Tx-100. To assess colocalization of PPARs and TH, after washing and blocking, the sections were first incubated overnight at 4 °C with the primary antibodies—anti-PPAR-α (1:100;11540-1-AP, Proteintech), anti-PPAR-β/δ (1:100;1056-2-AP, Proteintech), or anti-PPAR-γ (1:100;16643-1-AP, Proteintech), and anti-TH (AB152, EMD Millipore). For the secondary antibody-antigen reaction, the tissues were rinsed in PBS and then incubated in Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1:300; Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse antibody (1:300; Invitrogen) for 1 h at room temperature. The quantification of PPAR positive cells was performed while using the Olympus BX-51 microscope. PPAR isotype and TH dual-labeled cells were quantified in the SN and VTA.
9
MDSC Regulation of 4T1 Cell EMT
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4T1 cells were trypsinized, resuspended in culture media, co-cultured with MDSCs, and grew on round cover slips in 24-well plates. Trans-wells were incubated at 37°C containing 1:1, 1:3, 1:5, 1:10, and 1:20 cell number ratio of pre-isolated MDSC cells for 96 h. The morphology of 4T1 cells co-cultured with MDSCs for 24, 48, and 96 h were observed under white light microscope. To explore indirectly the role of MDSCs on EMT of the primary tumor cells, MDSCs were loaded into the upper chambers of a 24 well trans-well insert. After removing non-migrating cells using cotton swabs, inserts were washed in PBS, fixed in 4% PFA (Paraformaldehyde) and prepared for the immunofluorescence assay. Then the cells were permeabilized with 0.1% Triton X-100 in PBS, and stained with mouse monoclonal antibodies directed against E-cadherin and Vimentin (Abcam). The secondary antibodies were Alexa Fluor 488-conjugated donkey-anti-rabbit antibody (Invitrogen, 1:500). DAPI (4',6-diamidino-2-phenylindole) (5 μg/ml) was also stained for 10 min. The images were visualized through a Leica TCS SP5 confocal microscope.
10
Immunohistochemical Labeling of GFP
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Free-floating sections were first washed with 0.1 M PBS, and then incubated in a blocking solution that consisted of 2.5% BSA, 0.2% Triton X-100 and 5% donkey serum in 0.1 M PBS at 4°C for 2.5 h to minimize the background stain. Thereafter, sections were incubated at 4°C overnight with a rabbit anti-GFP (Invitrogen, Carlsbad, CA, USA) that was diluted with the blocking solution. After the sections were rinsed with 0.1 M PBS (15 min × 3 times), the sections were incubated with an Alexa fluor 488-conjugated donkey anti-rabbit antibody (Invitrogen, Carlsbad, CA, USA) in a dilution of 1:600. After washing, sections were mounted on glass slides with 75% glycerol in PBS and coverslipped.
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