Paraffin sections of cerebral cortex tissues were prepared, and the expression levels of NLRP3 and TLR4 were detected by an immunofluorescence analysis. After dewaxing and rehydration, the sections were placed in citrate buffer, and the antigens were recovered by a thermally mediated method. The sections were then incubated with 3% BSA for 30 min (Sigma–Aldrich, St. Louis, USA). Then, the sections were incubated overnight at 4 °C with diluted primary antibodies against NLRP3 (MA5–23919, Thermo Fisher Scientific) and TLR4 (ab22048, Abcam, Cambridge, UK). On the second day, the sections were incubated with a FITC-labelled secondary antibody at room temperature for 60 min. DAPI was used to stain the nuclei. Finally, the sections were dehydrated and examined under an inverted fluorescence microscope.
Ma5 23919
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The MA5-23919 is a laboratory equipment product by Thermo Fisher Scientific. It is designed for specific laboratory applications, but a detailed description cannot be provided while maintaining an unbiased and factual approach. Further details on the core function and intended use of this product are not available.
Automatically generated - may contain errors
Lab products found in correlation
Ab6717, by Abcam (2 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Ab22048, by Abcam (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Wl01372, by Wanlei (1 mentions)
Wl01980, by Wanlei (1 mentions)
Wla004, by Wanlei (1 mentions)
Wl00196, by Wanlei (1 mentions)
Wla023, by Wanlei (1 mentions)
Wl00891, by Wanlei (1 mentions)
T1081, by Solarbio (1 mentions)
Wl03450, by Wanlei (1 mentions)
Wla003, by Wanlei (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
Ab78494, by Abcam (1 mentions)
Anti gapdh ma1 16757, by Thermo Fisher Scientific (1 mentions)
Bc3710, by Solarbio (1 mentions)
Pc0020, by Solarbio (1 mentions)
A27039, by Thermo Fisher Scientific (1 mentions)
Ab178847, by Abcam (1 mentions)
6 protocols using ma5 23919
1
Immunofluorescence Analysis of NLRP3 and TLR4
2
Protein Quantification and Western Blot Analysis
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Firstly, BCA assay kit (WLA004, Wanleibio, China) and different concentrations of SDS-PAGE gels (WLA013, Wanleibio, China) were used to quantify the proteins in the brain homogenates or BV-2 cell lysates, which were then transferred to PVDF membranes (IPVH00010, Millipore, USA). Secondly, membranes were blocked with 5% non-fat dry milk (Q/NYLB 0039S, Yili, China) in TBST (T1081, Solarbio, China) and incubated with primary antibodies against galectin-3 (1:500, 60207-1-Ig, Proteintech, China), toll-like receptor 4 (TLR4, 1:400, WL00196, Wanleibio, China), nuclear factor kappa-B (NF-ĸBp65, 1:500, WL01980, Wanleibio, China), caspase-1 (1:500, WL03450, Wanleibio, China), interleukin-1β (IL-1β, 1:1000, WL00891, Wanleibio, China), NLRP3 (1:1000, MA5-23919, Thermo Fisher, USA), and β-actin (1:400, WL01372, Wanleibio, China), and then washed with TBST and incubated with HRP-IgG (1:5000, goat anti-rabbit, WLA023, Wanleibio, China). Lastly, ECL kit (WLA003, Wanleibio, China) was used to detect immunoreactive bands, and Gel-Pro-Analyzer software (WD-9413B, Beijing Liuyi, China) was used to scan and analyze bands. β-actin was set as internal standard to normalize film signals, and signals of sample in Control group were normalized to 1.0.
3
Immunofluorescence Assay for NLRP3 Detection
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Transfected cells were digested, counted, and incubated in IF chambers with 2×105 cells per well. When 60–80% confluence was reached, cells were washed with PBS (3×5 min) and fixed in 4% paraformaldehyde for 15 min, followed by additional PBS washing (3×5 min). Next, cells were permeabilized with 1% Triton X-100 in PBS (on ice, 2 min), washed with PBS (3×5 min), and then blocked in 5% serum for 1 h. After PBS washing (3×5 min), cells were incubated with primary antibody against NLRP3 (MA5-23919, Thermo Fisher Scientific), and then fluorescence labeled goat-anti-rabbit secondary antibody (ab6717, Abcam, Cambridge, UK) for 1 h. Afterwards, cells were stained with 4',6-diamidino-2-phenylindole (DAPI) for 15 min in the dark before observation under a fluorescence microscope (Olympus Corporation, Japan). Each assay was repeated thrice.
4
Immunofluorescence Analysis of KIM-1 and NLRP3
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After routine trypsinization, transfected HK-2 cells were counted and incubated in immunofluorescence chambers at 2 × 105 cells/well. After cell confluence reached 60 − 80%, the cells underwent three PBS rinses for 5 min each time and 15 min fixation with 4% paraformaldehyde. Following three PBS rinses for 5 min each time, the cells were permeabilized with 1% Triton X-100 (diluted in PBS) for 2 min on ice and rinsed with PBS 3 times for 5 min each time. The cells were blocked for 1 h in 5% serum, rinsed three times with PBS for 5 min each time, and probed with primary antibodies against KIM-1 (ab78494, 1:300, Abcam) and NLRP3 (MA5-23,919, 1:100, Thermo Fisher Scientific) prepared with PBS. Afterward, the cells were re-probed with green FITC fluorescence-labeled goat anti-rabbit secondary antibody (ab6717, 1:2000, Abcam) for 1 h at room temperature in the dark. Subsequent to 15 min nuclei staining with DAPI, the cells were observed and photographed using a fluorescence microscope under the same exposure conditions. Each experiment was repeated three times.
5
Protein Extraction and Western Blot Analysis
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After the experiment, the cells were collected, digested with 0.25% trypsin, collected into EP tubes, and protease inhibitors were added. The whole protein extraction kit (BC3710, Solarbio Life Sciences, Beijing, China) obtained the cell protein solution, and the BCA method (PC0020, Solarbio Life Sciences, Beijing, China) was used to detect the protein concentration. SDS-PAGE electrophoresis was performed, the protein was transferred to PVDF membrane and it was blocked with 5% nonfat milk powder for 1 h at room temperature, GAPDH (anti-GAPDH (MA1-16757, Invitrogen) was used as the normalizing antibody, and the corresponding primary antibodies (anti-NLRP3 (MA5-23919, Invitrogen), anti-ASC (PA5-83948, Invitrogen), anti-Caspase-1 (2225S, CST), and anti-GSDMD (39754S, CST)) were added and incubated at 4°C overnight. Horseradish peroxidase-labeled secondary antibody was used, and ECL color was developed, protein ladders are used to mark target protein positions (26617, thermo scientific). The gray value of the target band was analyzed with a gel image processing system (Image J software).
6
Ischemic Penumbra Assessment via Immunohistochemistry
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The ischemic penumbra of cerebral cortex is detected in the MCAO/R and MCAO/R+MCP groups, while the cerebral cortex is detected in the Sham group. Firstly, after antigen retrieval, the sections were fixed, permeabilized, and incubated with primary antibodies Iba1 (1:200, ab178847, Abcam, UK), Galectin-3 (1:100, 60207-1-Ig, Proteintech, China), NLRP3 (1:100, MA5-23919, Invitrogen, China). Secondly, the sections were incubated with a Cy3-conjugated (1:200, A27039, Invitrogen, China), or FITC-conjugated (1:100, Ab6785, Abcam, UK) secondary antibody. Finally, diamidino-2-phenylindole (DAPI) was added to stain the nuclei. All the stained sections were viewed by microscope (BX53, Olympus, Japan) and photographed by microscope photographic system (DP73, Olympus, Japan). Density of double-labeled cells was reported as the average number of double labeled cells per square millimeter in each group.
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