After l -NAT treatment, the total protein was extracted from the BRL cells and the rat liver tissue using protein extraction buffer (RIPA Beyotime Biotechnology, Shanghai, China). The cells were washed twice with ice-cold PBS (pH 7.4) and RIPA buffer (Solarbio, Beijing, China). Proteins were subjected to SDS-PAGE with a 10% running gel and then transferred to a PVDF membrane. The samples were incubated with the following antibodies: anti-NF-κB (1:500, Bioss, Beijing, bs-0465R); anti-TLR-4 (1:500, Bioss, Beijing, bs-1021R); anti-RIP2 (1:500, Santa Cruz); and anti-GAPDH (1:2000, Proteintech, 10494-1-AP), respectively. The details for the analysis have been described previously (Huang et al. 2015 (link)). Western blot images were analyzed using ImageJ software (National Institutes of Health, Bethesda, MD). GAPDH was used as the internal reference protein.
Anti tlr4
Manufactured by Bioss Antibodies
Sourced in China
Anti-TLR4 is an antibody that binds to the Toll-like receptor 4 (TLR4) protein. TLR4 is a member of the Toll-like receptor family and plays a critical role in the innate immune response by recognizing pathogen-associated molecular patterns. The Anti-TLR4 antibody can be used in laboratory research applications to study the function and regulation of TLR4.
Automatically generated - may contain errors
Lab products found in correlation
Anti nf κb, by Bioss Antibodies (3 mentions)
Anti nlrp3 antibody, by Bioss Antibodies (2 mentions)
Anti asc antibody, by Bioss Antibodies (2 mentions)
Ripa buffer, by Solarbio (1 mentions)
Anti rip2, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Anti caspase 1, by ABclonal (1 mentions)
Anti caspase 3, by Wuhan Servicebio Technology (1 mentions)
Anti myd88, by Bioss Antibodies (1 mentions)
Anti bax, by ABclonal (1 mentions)
Anti bcl 2, by ABclonal (1 mentions)
Fv500, by Olympus (1 mentions)
Anti il 1β antibody, by Bioss Antibodies (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Gapdh, by Proteintech (1 mentions)
Anti il 1β antibody, by Proteintech (1 mentions)
Uric acid, by Merck Group (1 mentions)
Anti glut9, by Bioss Antibodies (1 mentions)
Allopurinol, by Merck Group (1 mentions)
Benzbromarone, by Merck Group (1 mentions)
5 protocols using anti tlr4
1
Quantitative Analysis of NF-κB, TLR-4, and RIP2 in L-NAT Treated Cells
2
Immunohistochemical Analysis of Apoptosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissues were embedded using paraffin and cut into 4-μm-thick sections (n = 3 animals per group, 6 sections per animal). Sections were deparaffinized and rehydrated, then placed in a box filled with citric acid antigenic repair buffer (pH 6.0) in a microwave oven for antigen retrieval. Later, they were incubated with 3% H2O2 for 10 min at room temperature. After blocking with 5% BSA for 30 min, the tissue sections were incubated with primary antibody anti-Caspase1 (ABclonal, Wuhan, China; Catalog: A0964, diluted at 1:200), anti-Caspase3 (Servicebio, Wuhan, China; Catalog: GB11009-1, diluted at 1:200), anti-TLR4 (Bioss, Beijing, China; Catalogue: bs-20594R, diluted at 1:200), anti-MYD88 (Bioss, Beijing, China; Catalogue: bs-1047R, diluted at 1:200), anti-BAX (ABclonal, Wuhan, China; Catalog: A0207, diluted at 1:500), and anti-BCL-2 (ABclonal, Wuhan, China; Catalog: A0208, diluted at 1:500). The secondary antibody was incubated at room temperature. After PBS washing, the sections were monitored and captured under a microscope.
3
Immunofluorescence Analysis of Inflammasome Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BRL cells were exposed to H2O2 with or without L-NAT, fixed in 4% paraformaldehyde for 15 min and permeabilized with 0.3% Triton X-100-PBS for 15 min. Blocking was done in 5% goat serum for 30 min at 37 °C to inactivate endogenous peroxidase. Subsequent, the cells were incubated with anti-ASC antibody (1:200; Bioss), anti-NLRP3 antibody (1:200; Bioss), anti-IL-1β antibody (1:200; Bioss), anti-Caspase-1 antibody (1:100; Santa), anti-TLR4 (1:500; Bioss), and anti-NF-κB (1:500; Bioss) at 4 °C overnight and then incubated with FITC-conjugated secondary antibodies (1:150) at 37 °C for 30 min. DAPI staining was performed and image were taken using confocal microscopy (Olympus FV500; Olympus, Tokyo, Japan). Liver tissue from the sham group, I/R group and I/R + L-NAT group were made frozen sections of 10 µm thickness. And then immunofluorescence staining was performed using the method described above.
4
Quantitative Protein Analysis in Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For protein extraction, each of 100 mg liver tissues were homogenized on ice in 1 ml RIPA lysis buffer with protease inhibitor mix PMSF. Lysates were obtained by centrifugation at 15,000 rpm for 15 min at 4 °C. The protein concentration was quantified by BCA method. Subsequently, protein samples were subjected to 10% SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane, and then incubated with the following antibodies: anti-ASC antibody (1:200; Bioss), anti-NLRP3 antibody (1:200; Bioss), anti-TLR4 (1:500; Bioss) and anti-NF-κB (1:500; Bioss). Finally, the bands were observed using chemiluminescence (ECL) system according to the manufacturer’s instructions. Western blotting bands were analyzed using optical density scanning and Image Lab software (Bio-Rad). GAPDH (1:1000; Proteintech) was used as the loading control.
5
Uric Acid Regulation via Inflammatory Pathways
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Uric acid, benzbromarone, allopurinol and PO were purchased from Sigma (St. Louis, USA). Elisa kits for IL-1β and TNF-α were purchased from Xinbosheng (Shenzhen, China). Anti-GLUT9, anti-TLR4 and anti-NLPR3 antibodies were purchased from Bioss (Beijing, China). Anti-OAT1, anti-β-actin, anti-URAT1, anti-Anti-MyD88 and anti-IL-1β antibodies were purchased from Proteintech (Chicago, USA). Xanthine oxidase, urea nitrogen and adenosine deaminase kits were purchased from Jiancheng (Nanjing, China).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!