After treatments, kidney slides and podocyte culture coverslips (cell density is 1000/well in 24-well-plate) were fixed, blocked and incubated with primary antibodies against NLRP3 (1:100, Abcam, Cambridge, MA, USA), cleaved-caspase-1 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), VPS16 (1:100, Proteintech Group, Chicago, IL, USA), IL-1β (1:200, R&D systems, USA), Lamp-1(1:100, Abcam, Cambridge, MA, USA) at 4 °C overnight. Then samples were incubated with corresponding secondary antibodies with either Alexa- 488- or Alexa-555-labeled (Invitrogen) for 1 h at room temperature in the dark room. Finally, samples were mounted with mounting medium containing DAPI sealed with nail polish and pictures were taken under confocal laser scanning microscope (Fluoview FV1000; Olympus, Tokyo, Japan). There were 5–6 slides in every group, and 3 to 5 frames were chosen in every sample at random to show the characteristics of cell statues. Co-localization coefficient was analyzed with Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD) and expressed as Pearson’s correlation coefficient (PCC) [26 (link)].
Alexa 555 labeled
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa-555-labeled is a fluorescent dye conjugate that can be used for labeling and detection of biomolecules in various research applications. It consists of the Alexa Fluor 555 fluorophore covalently attached to a reactive group, allowing it to be incorporated into proteins, nucleic acids, or other molecules of interest.
Automatically generated - may contain errors
Lab products found in correlation
Alexa 488, by Thermo Fisher Scientific (5 mentions)
Cleaved caspase 1, by Santa Cruz Biotechnology (3 mentions)
Nlrp3, by Abcam (3 mentions)
Fluoview fv1000, by Olympus (3 mentions)
Vps16, by Proteintech (2 mentions)
Lamp1, by Abcam (2 mentions)
Il 1β, by R&D Systems (2 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Ab63856, by Abcam (2 mentions)
Ab14106, by Abcam (2 mentions)
Ab216130, by Abcam (2 mentions)
Ab23981, by Abcam (2 mentions)
Ab25245, by Abcam (2 mentions)
Cholecalciferol, by Merck Group (2 mentions)
Ab137029, by Abcam (2 mentions)
Alizarin red s solution tms 008 c, by Merck Group (2 mentions)
Desmin, by Thermo Fisher Scientific (1 mentions)
Podocin, by Merck Group (1 mentions)
Ab41803, by Santa Cruz Biotechnology (1 mentions)
Ab150687, by Abcam (1 mentions)
5 protocols using alexa 555 labeled
1
Immunofluorescence Analysis of Podocyte Markers
2
Immunofluorescence Analysis of Podocyte Markers
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After treatments, kidney slides and podocyte culture coverslips (cell density is 1000/well in 24-well-plate) were fixed, blocked and incubated with primary antibodies against NLRP3 (1:100, Abcam, Cambridge, MA, USA), cleaved-caspase-1 (1:200, Santa Cruz Biotechnology, Dallas, TX, USA), VPS16 (1:100, Proteintech Group, Chicago, IL, USA), IL-1β (1:200, R&D systems, USA), Lamp-1(1:100, Abcam, Cambridge, MA, USA) at 4 °C overnight. Then samples were incubated with corresponding secondary antibodies with either Alexa- 488- or Alexa-555-labeled (Invitrogen) for 1 h at room temperature in the dark room. Finally, samples were mounted with mounting medium containing DAPI sealed with nail polish and pictures were taken under confocal laser scanning microscope (Fluoview FV1000; Olympus, Tokyo, Japan). There were 5–6 slides in every group, and 3 to 5 frames were chosen in every sample at random to show the characteristics of cell statues. Co-localization coefficient was analyzed with Image Pro Plus 6.0 software (Media Cybernetics, Bethesda, MD) and expressed as Pearson’s correlation coefficient (PCC) [26 (link)].
3
Immunofluorescence Analysis of NLRP3 Inflammasome
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After treatments, kidney slides and podocyte culture coverslips were fixed, blocked and incubated with primary antibodies against NLRP3 (1:100, Abcam, Cambridge, MA, United States), ASC (1:200, Santa Cruz Biotechnology, Dallas, TX, United States), cleaved-caspase-1 (1:200, Santa Cruz Biotechnology, Dallas, TX, United States), podocin (1:400, Sigma), or desmin (1:400, Thermo Fisher Scientific) at 4°C overnight. Then slides were incubated with corresponding second antibodies with either Alexa- 488- or Alexa-555-labeled (Invitrogen, Carlsbad, CA, United States). For example, slides incubated with NLRP3 were then incubated with donkey anti goat secondary antibody, Alexa fluor plus 488, slides incubated with ASC, cleaved-caspase-1 or podocin were then incubated with donkey anti mouse secondary antibody, Alexa fluor plus 555, slides incubated with desmin were then incubated with donkey anti rabbit secondary antibody, Alexa fluor plus 488. After that, slides were observed with a laser scanning confocal microscope (Fluoview FV1000, Olympus, Japan). Co-localization coefficient was analyzed with Image Pro Plus 6.0 software and presented by Pearson’s correlation coefficient (PCC).
4
Osteogenic Differentiation and Mineralization Assays
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Cholecalciferol (vitamin D3) (C9756, Sigma Aldrich, St. Louis, MO, USA). Mouse monoclonal antibody against α-SMA (ab7817, Abcam, USA), rabbit polyclonal antibodies against acid ceramidase-α (ACα) (sc-292176, Santa cruz, USA), RUNX2 (ab23981, Abcam, USA), OSP (ab63856, Abcam, USA), SM22-α (ab14106, Abcam, USA), VPS16 (Cat. No.17776-1-AP, Protein biotech group, USA), Rab7 (ab137029, Abcam, USA), CD63 (ab216130, Abcam, USA), annexin-II (AnX2, ab41803, USA), and alkaline phosphatase (ALP, sc-28904, Santa Cruz, USA). Rat monoclonal anti-mouse Lamp-1 (ab25245, Abcam, USA), Cre (Cat No. 6905, Novagen EMD Millipore, Billerica MA, USA), and ceramide (MID 15B4, Enzo ALX-804-196-T050). Secondary antibodies are Alexa-488 or Alexa-555-labeled (Life technologies, USA). Von Kossa staining kit (ab150687, Abcam, USA) and Alizarin Red S Solution (TMS-008-C, EMD Millipore. USA) were used for detection AMC.
5
Arterial Medial Calcification: Immunochemistry
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Cholecalciferol (vitamin D3) (C9756, Sigma Aldrich, St. Louis, MO, USA). Mucolipin1 (SC-26269, Dallas, TX, USA), OSP (ab63856, Abcam, Cambridge, MA, USA), RUNX2 (ab23981, Abcam, Cambridge, MA, USA), SM22-α (ab14106, Abcam, Cambridge, MA, USA), VPS16 (Cat. No.17776–1-AP, Protein biotech group, Rosemont, IL, USA), Rab7 (ab137029, Abcam, Cambridge, MA, USA), ALG-2 (PDCD6, A6685, Cummings Park Dr, Woburn, MA, USA.), CD63 (ab216130, Abcam, Cambridge, MA, USA) and annexin-II (AnX2, ab41803, Abcam, Cambridge, MA, USA). Secondary antibodies are Alexa-488 or Alexa-555-labeled (Life technologies, Grand Island, NY, USA). Rat monoclonal anti-mouse Lamp-1 (ab25245, Abcam, Cambridge, MA, USA). Alizarin Red S Solution (TMS-008-C, EMD Millipore. Burlington, MA, USA) was used for detection of arterial medial calcification.
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