HCEC cultures on 8-chamber slides and mouse eye sections were fixed with freshly prepared 2% paraformaldehyde at 4°C for 10 minutes. Cell cultures were permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. Indirect immunofluorescent and immunohistochemical staining was performed using our previous methods [32 (link), 33 (link)]. Primary rabbit polyclonal antibodies against human and mouse NLRP3 (NBP1-77080), caspase-1 (NB100-56565), caspase 8 (NB100-56116, Novus Biologicals, Littleton, CO), NLRP6 (PA5-21022), IL-1β (P420B), BRCC36 (PA5-20426, Thermo Scientific, Rochford, IL), ASC (N-15, SC-22514R), IL-18 (SC-6179, Santa Cruz Biotechnology, Dallas, TX), aconitase-2 (ab129105), 8-OHdG (ab62623, Abcam, Cambridge, MA) were used. Alexa-Fluor 488 conjugated secondary antibodies (R&D Systems) were applied, and propidium iodide (PI) or 4′, 6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Secondary antibodies were applied alone were as a negative control and compared to isotype goat IgG. The staining will be photographed with Zeiss laser scanning confocal microscope (LSCM510META, Thornwood, NY).
Alexa fluor 488 conjugated secondary antibodies
Manufactured by R&D Systems
Alexa Fluor 488 conjugated secondary antibodies are fluorescent probes used to detect and visualize target proteins in various applications, such as immunohistochemistry, flow cytometry, and Western blotting. These antibodies are conjugated with the Alexa Fluor 488 dye, which emits green fluorescence when excited by light at the appropriate wavelength. The Alexa Fluor 488 dye provides bright, photostable fluorescence, making it a useful tool for protein detection and localization studies.
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2 protocols using alexa fluor 488 conjugated secondary antibodies
1
Immunostaining of NLRP3 and Inflammasome Proteins
2
Immunofluorescent Staining of Dectin-1 and PGLYRPs
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The human corneal epithelial cells were fixed with freshly prepared 2% paraformaldehyde at 4°C for 10 minutes. Cell cultures were permeabilized with 0.2% Triton X-100 in PBS at room temperature for 10 min. Indirect immunofluorescent staining was performed as previously described [35 (link),36 (link)]. Primary goat polyclonal antibody against human dectin-1 and PGLYRP-2 (SC-50472, Santa Cruz Biotechnology, Dallas, TX), PGLYRP-3 (IMG-391), PGLYRP-4 (MG-414, Imgenex, San Diego, CA), and p65 (622602, BioLegend) were used. Alexa-Fluor 488 conjugated secondary antibodies (R&D Systems) were applied, and propidium iodide (PI) was used for nuclear counterstaining. Secondary antibodies were applied alone were as a negative control and compared to isotype goat IgG. Imaging was completed with a Zeiss laser scanning confocal microscope (LSCM510META, Thornwood, NY).
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