Western blotting was routinely performed as previously reported [20 (link)]. Mouse anti-β-actin (1:10,000, Proteintech, Chicago, IL), rabbit anti-NLRP3 (1:500, Novus Biologicals, CO, USA), rabbit anti-caspase-1 (1:500, Proteintech), mouse anti-ASC (1:500, Santa Cruz, CA, USA), rabbit anti-GSDMD (1:1000, CST, Danvers, USA), mouse anti-pro-IL-1β (1:1000, Proteintech), rabbit anti-cleaved IL-1β (1:1000, Novus Biologicals), rabbit anti-pro-IL-18 (1:1000, Proteintech), rabbit anti-cleaved IL-18 (1:300, Bioss, Beijing, China), and rabbit anti-caspase-11 (1:1000, Novus Biologicals) were used. The densities of protein blots were quantified by using ImageJ software (NIH, Bethesda, MD) and normalized to the level of β-actin.
Rabbit anti nlrp3
Manufactured by Novus Biologicals
Sourced in United States, United Kingdom
Rabbit anti-NLRP3 is a primary antibody that recognizes the NLRP3 protein. NLRP3 is a member of the NOD-like receptor (NLR) family and plays a key role in the formation of the NLRP3 inflammasome, which is involved in the regulation of inflammatory responses.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti β actin, by Proteintech (2 mentions)
Rabbit anti caspase 1, by Proteintech (2 mentions)
Mouse anti asc, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti iba1, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Alexa fluor 594 conjugated goat anti rabbit igg, by Abcam (1 mentions)
Rabbit anti neun, by Proteintech (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Solarbio (1 mentions)
Fluorescence microscope, by Nikon (1 mentions)
Rabbit anti il 1β, by Abcam (1 mentions)
Horseradish peroxidase conjugated anti rabbit secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti pro caspase 1, by Abcam (1 mentions)
Rabbit anti gapdh, by Proteintech (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
4 protocols using rabbit anti nlrp3
1
Western Blot Analysis of Inflammasome Proteins
2
Immunofluorescence Analysis of Neuroinflammation
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All mice were sacrificed after behavior tests. Mice brains were fixed with 4% paraformaldehyde, and brain slices (4 μm thick) were prepared and used for staining. The slices were washed in PBS for 5 min three times and then blotted in 5% goat serum for 1 h at room temperature. The slices were incubated with the primary antibodies overnight at 4°C. The primary antibodies used were: rabbit anti-IBA-1 (1:200, Abcam, United Kingdom), rabbit anti-NLRP3 (1:200, Novus Biologicals, United States), rabbit anti-caspase-1, rabbit anti-GSDMD-N, and rabbit anti-NeuN (1:100, Proteintech, China). After being washed in PBS three times, these slices were incubated with secondary antibodies at 37°C for 1 h, and the secondary antibodies used were Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:400, Abcam, United Kingdom) and Alexa Fluor 594-conjugated goat anti-rabbit IgG (1:400, Abcam, United States). Then, these slices were washed three times again and stained with DAPI (Solarbio, China) for 5 min. The results were imaged using a fluorescence microscope (Nikon, Japan).
3
Quantification of Apoptosis Markers
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Western blot analysis was performed as previously reported (Kristian et al., 2011). The protein extracted from the left cortex was used for western blot, and equal amounts of protein (50 μg) were loaded in each lane of the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel. After electrophoresis, the samples were transferred onto a nitrocellulose membrane. Then, the membrane was blocked for 2 hours at room temperature using blocking buffer and incubated at 4°C overnight with the following primary antibodies: rabbit anti-cleaved caspase-8 (1:1000, Abcam), rabbit anti-NLRP3 (1:500, NOVUS, St. Louis, MO, USA), rabbit anti-cleaved caspase-1 (1:1000, NOVUSA), and rabbit anti-IL-1β (1:1000, Abcam). Rabbit anti-beta-actin was used as an internal loading control (1:6000, Santa Cruz Biotechnology). The bands were quantified using densitometry with ImageJ 1.4 software (National Institutes of Health, Bethesda, MD, USA). The membranes were washed with Tris-buffered saline-Tween 20 for 30 minutes, and then incubated with horseradish peroxidase-conjugated secondary anti-rabbit antibody (1:4000, Santa Cruz Biotechnology) at room temperature for 2 hours.
4
Western Blot Analysis of Protein Expression
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Western blotting was routinely performed as previously reported (34) . Rodent brain tissues, BV2 cells, and RAW264.7 cells were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China) containing protease inhibitor cocktail. After denatured in loading buffer, the samples were subjected to SDS-PAGE and then transferred to PVDF membranes (Millipore, MA, USA). After blocked by 5% non-fat milk, the membranes were incubated overnight at 4°C with the primary antibodies as below: Mouse anti-β-actin (Proteintech, IL, USA), rabbit anti-GAPDH (Proteintech), rabbit anti-TRPM4 (Sigma-Aldrich), mouse anti-SUR1 (Abcam, Cambridge, UK), rabbit anti-NLRP3 (Novus, CO, USA), rabbit anti-pro-caspase-1 (Abcam), rabbit anticaspase-1 p20 (Bioss, Beijing, China), rabbit anti-precursor of IL-1β (pro-IL-1β) (Proteintech), rabbit anti-IL-1β p17 (Novus), rabbit anti-Kir6.1 (Abcam), and rabbit anti-Kir6.2 (Abcam), followed by the detection via the secondary antibodies (CST, MA, USA). The densities of protein blots were quanti ed by ImageJ software (NIH, MD, USA) and normalized to the level of β-actin or GAPDH.
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