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).
Nb100 56116
Manufactured by Novus Biologicals
Sourced in United States
The NB100-56116 is a laboratory equipment product manufactured by Novus Biologicals. It is designed for general laboratory use, but a detailed unbiased description of its core function is not available.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 conjugated secondary antibodies, by R&D Systems (1 mentions)
Lscm 510 meta, by Zeiss (1 mentions)
Nbp1 77080, by Novus Biologicals (1 mentions)
Pa5 21022, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Cleaved caspase 8, by Novus Biologicals (1 mentions)
Supersignal west dura substrate, by Thermo Fisher Scientific (1 mentions)
P0447, by Agilent Technologies (1 mentions)
Tris hcl ready gel, by Bio-Rad (1 mentions)
Gapdh, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene fluoride membrane, by Bio-Rad (1 mentions)
Gill s hematoxylin no 3, by Merck Group (1 mentions)
Dako blocking reagent, by Agilent Technologies (1 mentions)
4 protocols using nb100 56116
1
Immunostaining of NLRP3 and Inflammasome Proteins
2
Necroptosis Signaling Pathway Analysis
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Anti-RIP3 (ab56164; 1:800), anti-MLKL (ab194699; 1:1000), anti-MLKL (phospho S358) (ab187091; 1:1000), anti-caspase-8 (NB100-56116; Novus Biologicals, Colorado, USA 1:1000), anti-CXCR2 (ab65968; 1:500), anti-CXCL5 (ab9802; 1:1000) and anti-β-actin (ab8227; 1:2000) antibodies and then probed with secondary antibodies conjugated to horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Immunoblots were detected by enhanced chemiluminescence with ChemiDocXRS (Bio-Rad Laboratories). Each experiment was repeated more than three times.
3
Western Blot Analysis of Cleaved Caspase 8
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Proteins were separated by 4% to 15% SDS-PAGE (Tris-HCl Ready Gels; Bio-Rad Laboratories) and transferred to polyvinylidene fluoride membranes (Bio-Rad Laboratories). The membranes were incubated overnight with primary antibodies: cleaved caspase 8 (NB100-56116; Novus Biologicals, Littleton, CO, USA) and GAPDH (MA5-15738; Thermo Scientific, Rockford, IL, USA). Secondary polyclonal goat anti-immunoglobulin antibodies were from Dako (P0447, P0448; Glostrup, Denmark). Detection was by chemiluminescence substrate (SuperSignal West Dura Substrate; Thermo Scientific) according to the manufacturer's protocols. Quantitative densitometry of the immunoblots was performed using ImageJ software (http://rsb.info.nih.gov/ij/index.html , provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) and expressed as the mean density (±SD) from replicate experimental groups. All experiments were performed a minimum of three times.
4
Immunohistochemical Analysis of Regulated Cell Death
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For IHC, paraffin-embedded sections (3.5 μm) were dewaxed and rehydrated. Antigen retrieval was performed by incubation in sodium citrate buffer pH 6.0 at 65 °C overnight. After cooling, unspecific antigens were blocked with the DAKO blocking reagent (Dako, Glostrup, Denmark), followed by overnight incubation with the 1:250 diluted rabbit monoclonal antibody [EPR9514] against human p-MLKL (phospho S358; abcam, Cambridge, UK), human cleaved caspase 8 (NB100-56116; NOVUS Biologicals, Centennial, CO, USA) or RIPK3 (GTX107574; GeneTex, Irvine, CA, USA) at 4 °C. Sections were treated with 3% hydrogen peroxide before starting the staining with the Dako LSAB2 System-HRP kit (Dako, Glostrup, Denmark). In all samples a final staining of cell nuclei by Gill’s hematoxylin No 3 (Sigma-Aldrich) was performed. In case of cleaved CASP8 and p-MLKL, the percentage of positive cells was quantified by manual counting of the stained sections.
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