AIS primary chondrocytes after incubation with IL-6 were first washed with PBS, fixed with 4% paraformaldehyde for 25 min, incubated with 0.5% Triton X-100 for 10 min, and then blocked with 5% BSA in PBS for 30 min. Chondrocytes were then incubated overnight with primary antibody against Ki67 (1:500; # 9449; RRID: AB_2797703; CST) at 4 °C. After washing with PBS, the chondrocytes were incubated with goat anti-rabbit secondary antibody and Alexa Fluor 488-conjugated antibody (1:300; Sigma, USA) for 1 h. Finally, the cells were incubated with DAPI (Solarbio, China) for 5 min. Pictures were acquired by immunofluorescence microscopy (Leica, Germany). The results were detected quantitatively using ImageJ software (Bethesda, USA).
Alexafluor 488 conjugated antibody
Manufactured by Merck Group
Sourced in United States
AlexaFluor 488-conjugated antibody is a fluorescent-labeled antibody used as a detection reagent in various laboratory applications. It is designed to bind to and label specific target molecules, allowing their visualization and quantification through fluorescence detection techniques.
Automatically generated - may contain errors
Lab products found in correlation
Immunofluorescence microscopy, by Leica (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Operetta cls high content analysis system, by PerkinElmer (1 mentions)
Harmony software, by PerkinElmer (1 mentions)
Fibronectin, by Santa Cruz Biotechnology (1 mentions)
Snail1, by Santa Cruz Biotechnology (1 mentions)
Tritc conjugated antibody, by Merck Group (1 mentions)
E cadherin, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Mitoxantrone, by Merck Group (1 mentions)
Topotecan, by Merck Group (1 mentions)
Bovine serum, by Corning (1 mentions)
Sn 38, by Merck Group (1 mentions)
Az628, by Selleck Chemicals (1 mentions)
Trypsin, by Corning (1 mentions)
Hrp conjugated secondary antibody, by Merck Group (1 mentions)
Paclitaxel, by Merck Group (1 mentions)
Cisplatin, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
5 protocols using alexafluor 488 conjugated antibody
1
Chondrocyte Proliferation Assay with IL-6
2
Evaluating Anti-OROV Monoclonal Antibodies
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The reactivity of the anti-OROV 63B3E7 and 268B8A3 mAbs was evaluated by IFA in several cell lines infected by OROV (Vero E6, A549, Huh-7.5, and SH-SY5Y). Briefly, 2 × 104 cells/well seeded in 96-well plates were infected with OROV (MOI of 0.02) and incubated at 48 h. At the end of the incubation period, the cells were fixed with 200 µl/well of methanol-acetone (1:1) at -20°C for 1 h. Next, 100 µl/well of anti-OROV mAbs 63B3E7 and 268B8A3, diluted at 1:800 and 1:100, respectively, in 1% PBS-BSA, was incubated for 1 h at 37°C. The mAbs dilutions were determined based on the results showed in the Fig. 1 D. After three washes with PBS-T, a secondary anti-mouse IgG or IgM Alexa Fluor 488-conjugated antibody (1:1000; Sigma-Aldrich) and DAPI (0.3 mM; 4’,6-diamidino-2-phenylindole) diluted in 1% PBS-BSA were used to stain cells. The immunofluorescence images were acquired on an Operetta CLS High-Content Analysis System (PerkinElmer, Massachusetts, USA) using a 20x non-confocal objective and analyzed using the Harmony software (PerkinElmer).
3
Antibody Sourcing for Cellular Analysis
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Primary antibodies to E-cadherin, GAPDH, fibronectin, SNAIL-1 and TBP were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA); primary antibody to β-catenin was from Abcam (Cambridge, UK); primary antibody to vimentin was from Sigma; primary antibody to α-SMA was from GeneTex (Irving, CA). Secondary horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies were from goat (Bio-Rad Laboratories); AlexaFluor 488-conjugated antibody was from Millipore (Billerica, MA); TRITC-conjugated antibody was from Sigma.
4
Uptake and Efflux of Chemotherapeutics
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AZ-628 was purchased from Selleckchem (Houston, TX, United States). Chemotherapeutic agents including mitoxantrone, topotecan, SN-38, paclitaxel, and cisplatin were purchased from Sigma Co. (St. Louis, MO, United States). Cell culture reagents including bovine serum, fetal bovine serum, DMEM and 0.25% trypsin were purchased from Corning Inc. (Tewksbury, MA, United States). Immunoblotting materials including human ABCG2 monoclonal antibodies for ABCG2 and GAPDH, Alexa Fluor 488 conjugated antibody and HRP-conjugated secondary antibody were purchased from Millipore (Billerica, MA, United States). Tritium-labeled mitoxantrone ([3H]-mitoxantrone, 2.5 Ci/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA, United States).
5
Immunohistochemical Analysis of Brain Tissue and Cell Lines
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The deparaffinized slides of brain tissues were permeabilized with 0.1 M PBS, 5% FBS, and 0.3% Triton X for 1 h at room temperature. The sections were subsequently incubated with NeuN (1:200, ab177487, Abcam), GFAP (1:200, 3670, CST), SGLT1(1:200, 07-1417, Millipore) or SGLT2(1:200, ab85626, Abcam) antibodies at 4° C overnight. Subsequently, samples were incubated with an Alexa Fluor 488-conjugated antibody (diluted 1:200 for SGLT1 and SGLT2, Millipore) or Alexa Fluor 594-conjugated antibody (diluted 1:200 for NeuN and GFAP, Millipore) for 2 h at room temperature and were counterstained with DAPI for 10 min. All images were captured using a microscope (Olympus, Japan).
For HT-22 cells, the cells after treatment were subjected to incubation anti-SGLT1 primary antibody (07-1417, Millipore) at 4° C overnight. Other steps were the same as the brain sections.
For HT-22 cells, the cells after treatment were subjected to incubation anti-SGLT1 primary antibody (07-1417, Millipore) at 4° C overnight. Other steps were the same as the brain sections.
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