ABT-263 (Navitoclax), 3-MA (3-methyladenine) and Rapamycin (Sirolimus) were purchased from Selleck Chemicals (TX, USA). The primary Rabbit monoclonal antibodies of Bcl-2, Bax, Beclin-1(Becn), Atg5, LC-3, Trem-2, and Tubulin-α conjugated with Alexa Fluor® 790 were acquired from Abcam (Cambridge, UK). The secondary antibodies Goat anti-Rabbit IgG H&L (Alexa Fluor®680) was purchased from Abcam as well. The DAPI (4′, 6-diamidino-2- phenylindole) was obtained from Beyotime (Shanghai, China). The F4/80-PE anti-mouse antibody, PE Rat IgG2a isotype and biotin anti-mouse CD16/32 antibody were purchased from Biolegend (San Diego, CA). The Phallodin-iFluor 633 Conjugate was purchased from ATT Bioquest® (Sunnyvale, CA). The cell culture reagents, RPMI 1640 and FBS (fetal bovine serum) were purchased from Biological Industries (Cromwell, CT). The RT2 First Strand kit and Profiler PCR Array (PAXX-084) and SYBR Green qPCR master mix were purchased from Qiagen (Hilden, Germany). Proteome Profiler Array of Mouse cytokine (ARY006) was purchased from R&D systems, Inc. (MN, USA).
Alexa fluor 790
Alexa Fluor® 790 is a fluorescent dye. It has an excitation maximum at 784 nm and an emission maximum at 814 nm.
Lab products found in correlation
6 protocols using alexa fluor 790
Investigating Autophagy and Inflammation Pathways
Protein Quantification and Western Blotting
Western Blot Analysis of Larval Brain
Western Blot Analysis of Epithelial-Mesenchymal Transition
Western Blot Analysis of Autophagy Markers
Western Blot Analysis of Autophagy and Apoptosis Markers
The total protein of the cells or spleen tissue were extracted using 1% Triton X-100 (Beyotime) with 1x protease inhibitor cocktail (Beyotime), and then the concentration of protein was quantified using the Bradford protein assay kit (Beyotime). Twenty micrograms of protein was separated by SDS-PAGE and transferred to a PVDF membrane (0.22 μm, Bio-Rad, USA). After blocking with QuickBlock buffer (Beyotime), the membranes were incubated and shaking gently overnight at 4 °C with the primary antibody against LC-3 (1:2000), Bcl-2 (1:2000), Bax (1:2000), Beclin-1 (Becn; 1:2000), Atg5 (1:2000), Trem-2 (1:1000). All the primary and secondary antibodies were purchase from Abcam Inc.(UK). After washing with TBS-T extensively, the PVDF membranes were incubated with an appropriated Alexa Fluor® 680-conjugated secondary antibody (1:10000) for 1 h at room temperature on the next day. The bands were detected and analyzed with Odyssey CLx (LI-COR, USA) system. After the detection the PVDF membranes were stripped and incubated with Tubulin-α conjugated with Alexa Fluor® 790 (1:10000, Abcam, UK) for 2 h at room temperature, and detected the band for the loading control to normalized the interested proteins. The intensity of these bands were quantified with Image Studio software (LI-COR, version 5.2.5).
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