Cells (106 cells/well) were sonicated in an ice bath, lysed with lysis solution containing protease inhibitors and then centrifuged at 4 °C and 12,000 rpm for 15 min. The total protein concentration was determined by a BCA protein concentration assay kit (MD913053; MDL, Beijing, China). The proteins were separated by SDS–PAGE (10%) and transferred onto polyvinylidene difluoride (PVDF) membranes (ISEQ00010, 0.22 μm; Millipore, Billerica, MA, USA) by transfer electrophoresis. The membrane was blocked with nonfat dry milk (5%) in Tris-buffered saline–Tween (TBST) at room temperature for 1 h. The proteins on the membrane were reacted with the corresponding primary antibodies against IκBα, phospho-IκBα, p65, phospho-p65, CD36 (bs-1287R, bs-2513R, bs-0465R, bs-0982R, bs-1100R; Bioss, Beijing, China) and β-actin (MD6553; MDL, Beijing, China) overnight at 4 °C. The membrane was incubated with the anti-rabbit secondary antibodies (1:4000; MD912577; MDL, Beijing, China) at room temperature for 1 h in the dark after being washed with TBST. The membrane was washed with TBST again, and finally, the protein bands were captured with a chemiluminescence imaging system (ChemiScope 6100, CLINX Science Instruments, Shanghai, China).
Bs 0465r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-0465R is a precision laboratory equipment designed for accurate and reproducible liquid handling. It features an adjustable pipette volume range and can accommodate a variety of sample sizes. The device is constructed with high-quality materials to ensure reliable performance and durability in the laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Bs 0982r, by Bioss Antibodies (3 mentions)
Bs 2513r, by Bioss Antibodies (2 mentions)
Bs 0061r, by Bioss Antibodies (2 mentions)
Bs 1287r, by Bioss Antibodies (1 mentions)
Bs 1100r, by Bioss Antibodies (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Chemiscope 6100, by Clinx (1 mentions)
G1211, by Wuhan Servicebio Technology (1 mentions)
Gb11519, by Wuhan Servicebio Technology (1 mentions)
Ab47363, by Abcam (1 mentions)
Ab170099, by Abcam (1 mentions)
Bs 2637r, by Bioss Antibodies (1 mentions)
A16288, by ABclonal (1 mentions)
Bs 17591r, by Bioss Antibodies (1 mentions)
Bsm 33207m, by Bioss Antibodies (1 mentions)
Ecl reagent, by Beyotime (1 mentions)
Ab85309, by Abcam (1 mentions)
Ac026, by ABclonal (1 mentions)
Bs 5476r, by Bioss Antibodies (1 mentions)
Bs 0637r, by Bioss Antibodies (1 mentions)
11 protocols using bs 0465r
1
Western Blot Analysis of Protein Signaling
2
Immunohistochemical Analysis of TLR4, MyD88, and NF-κB p65
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical analysis was performed using anti-TLR4 (1:1000, GB11519, Servicebio), anti-MyD88 (1:200, GB11269, Servicebio), and anti-NF-κB p65 (1:400, bs-0465R, Bioss) antibodies. Fixed tissue gradient alcohol dehydration, paraffin embedding, and slicing to a thickness of 4 μm were performed. After deparaffinization and rehydration, the tissue sections were antigen-repaired with citric acid antigen-repair buffer (pH 6.0) and heated for the specified time. After natural cooling, the slides were placed in PBS (pH 7.4) and washed by shaking on a decolorization shaker three times for 5 min each. After treatment with 3% H2O2 and incubation at room temperature in the dark for 25 min, the slides were placed in PBS (pH 7.4) and washed by shaking on a decolorization shaker 3 times for 5 min each. Subsequently, 3% BSA was added to the circle to cover the tissue evenly, and the tissues were sealed for 30 min at room temperature. After gently removing the sealing solution, the sections were placed flat in a wet box with primary antibodies and incubated overnight at 4°C, followed by incubation with secondary antibodies for 30 min. The sections were then stained with diaminobenzidine (DAB) (G1211, Servicebio) and hematoxylin and mounted with neutral balsam. Brownish-yellow granules indicated positive signals. The slides were scanned and evaluated using Image-Pro Plus software.
3
Western Blot for Protein Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The detailed process of Western blot was described according to our previous study (18 (link)). The primary antibodies included NF-κB (bs-0465R, 1:1000, Bioss), p-NFκB (#3033, 1:1000, CST), p38 (ab170099, 1:1000, abcam), p-p38 (ab47363, 1:500, abcam), JNK (#9252, 1:1000, CST), p-JNK (#4668, 1:1000, CST), and β-actin (66009-1-Ig, 1:5000, Proteintech).
4
Protein Expression Analysis in Tracheal Tissue and Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was used to measure the related proteins. The total protein of tracheal tissue and HD11 cells was extracted by the whole-cell lysis method. Primary antibodies for TLR4 (bs-20379R, Bioss, Beijing, China), IκBα (10268-1-AP, Proteintech, Wuhan, China), p-IκBα (bs-2513R, Bioss), p65 (bs-0465R, Bioss), p-p65 (bs-0982R, Bioss), IL-1β (A16288, ABclonal, Wuhan, China), TNF-α (bsm-33207M, Bioss), JNK (bs-20760R, Bioss), p-JNK (bs-17591R, Bioss), ERK (bs-2637R, Bioss), p-ERK (bs-1645R, Bioss), and GAPDH (A5028, bimake, Houston, TX) (all at 1:1,000 dilution) protein were incubated for 12 h at 4°C. Secondary anti-rabbit IgG horseradish peroxidases (bs-0061R, Bioss) were incubated for 1.5 h. Enhanced chemiluminescence (ECL ) reagent (Beyotime, China) was used to visualize the bound immune complexes by automatic chemiluminescence image analysis system (Tanon, China) and the density of the protein bands was measured with Image J software (V 1.42, National Institutes of Health; Hu et al., 2021 ).
5
Protein Expression Analysis in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total protein was extracted from the cells using RIPA lysis buffer, and the protein concentration was determined using the BCA assay kit. After denaturation at 95 °C, the protein was stored at -80 °C. Equal amounts of protein were loaded to each lane and separated by 10% SDS-PAGE. The separated proteins were then transferred onto PVDF membranes and blocked with 5% skim milk powder for 2 h. The PVDF membranes were then incubated overnight at 4 °C with appropriate primary antibodies against β-actin (AC026, Abclonal, 1:50,000), Sirt1 (60303-1-lg, Proteintech, 1:2000), AP-1 (66313-1-Ig, Proteintech, 1:2000), NF-κB p65 (bs-0465R, Bioss, 1:2000), NF-κB p-p65 (82335-1-RR, Proteintech, 1:2000), p-P38 (bs-5476R, Bioss, 1:2000), P38 (bs-0637R, Bioss, 1:2000), p-JNK (80024-1-RR, Proteintech, 1:2000), JNK (66210-1-lg, Proteintech, 1:2000), HO-1 (ab85309, Abcam, 1:3000), and Nrf2 (16396-1-AP, Proteintech, 1:2000). After washing with TBST, the membranes were incubated at room temperature for 2 h with Goat Anti-Rabbit IgG (H + L) HRP (S0001, Affbiotech, 1:5000), followed by detection using enhanced chemiluminescence (ECL). The bands were exposed using the fluorescence imaging system V2.0 (Tanon, Shanghai, China), scanned, and analyzed using Gel-Pro analyzer 4.0. The relative expression levels of the target proteins were calculated with β-actin as an internal reference.
6
Western Blot Analysis of Cardiac Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein samples (50 mg) were separated in SDS-PAGE and transferred onto a nitrocellulose membrane. The blots were blocked with 5% nonfat milk for 2 h at room temperature, then incubated with primary antibody including BNP (1:1,000 dilution, sc-271185; Santa Cruz), β-MHC (1:2,000 dilution, SAB2106550; Sigma), Beclin-1 (1:1,000 dilution, ab207612; Abcam), LC3-II/I (1:500 dilution, GTX100240; GeneTex), p62 (1:1,000 dilution, 5114S; CST), P-AMPK (1:500 dilution, bs-4002R; Bioss), T-AMPK (1:1,000 dilution, E-AB-33742; Elabscience), P-mTOR (1:500 dilution, bs-3494R; Bioss), T-mTOR (1:200 dilution, BM4182; Boster), PPARγ (1:500 dilution, bs-4590R; Bioss), Nuclear NF-κB (1:500 dilution, bs-0465R; Bioss), total NF-κB (1:200 dilution; Abcam), GAPDH (1:2000; Zhong Shan-Golden Bridge Biological Technology), and β-actin (1:5,000 dilution; Santa Cruz), respectively, at 4°C overnight. The western blot bands were collected by Imaging System (LI-COR Biosciences) and quantified with Odyssey v.1.2 software by measuring intensity (area × optical density [OD]) in each group with β-actin as the internal control.
7
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The protein lysate was obtained from tissue by culture cell total protein extraction reagent (BOSTER, Wuhan, CHN) and mammalian tissue protein extraction reagent (BOSTER, Wuhan, CHN), with protease inhibitor and phosphatase inhibitor cocktail, respectively. Protein concentration was determined by a BCA protein analysis kit (BIOMED, Beijing, CHN). The primary antibodies included the following: rabbit anti-β-actin (#20536-1-AP; Proteintech, Wuhan, CHN), rabbit anti-ZO-1 (#21773-1-AP; Proteintech, Wuhan, CHN), rabbit anti-occludin (#27260-1-AP; Proteintech, Wuhan, CHN), rabbit anti-Claudin 1 (#bs-1428R; Bioss, Beijing, CHN), rabbit anti-NF-κB P65 (#bs-0465R; Bioss, Beijing, CHN), rabbit anti-phospho-NF-κB p65 (#3033T; CST, Danvers, MA USA), rabbit anti-p38 (#ab27986; Abcam, Cambridge, MA, USA), and rabbit anti-phospho-p38(#ab47363; Abcam, Cambridge, MA, USA). The secondary antibody used was goat anti-rabbit (#bs-0296G; Bioss, Beijing, CHN).
8
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The nucleoproteins were extracted using the Nucleoprotein Extracted Kit (Bestbio, Shanghai, China), according to the manufacturer’s instructions. Total protein was extracted using the following conventional method. Tissues were lysed in RIPA buffer (50 mM Tris-Cl pH7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate) containing 1× complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA). The samples were vigorously vortexed for 15 seconds and centrifuged at 10,000 g for 15 minutes at 4 ℃. Total protein (40 µg) was resolved on 8% sodium dodecyl sulfate polyacrylamide gels and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked for 30 minutes with 5% nonfat milk in phosphate buffered saline Tween (PBST; 0.1% Tween 20 in PBS) at room temperature, incubated with specific primary antibodies at 4 ℃ overnight, washed 3 times with PBST, and incubated with the appropriate secondary horseradish peroxidase-conjugated antibody (Pierce Antibody, Thermo Scientific, Shanghai, China) for 60 minutes at room temperature. The primary antibodies used were p65 (bs-0465R; Bioss, China), lamin B (ab8982; Abcam), and β-actin (ab8227; Abcam). The specific protein bands were detected with Immun-StarTM Western Chemiluminescence kit (Bio-Rad).
9
Quantifying NLRP3 and NF-κB Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chicken lung tissues from each group was immersed in protein lysate and centrifuged, and supernatant was removed. The protein concentration was measured using the BCA detection kit. The same amount of protein was subjected to electrophoresis on polyacrylamide gel and transferred to a PVDF membrane using wet transformation method (110 V, 90 min). The membrane was blocked with 5% fetal bovine serum and incubated with primary NLRP3 polyclonal antibody (1:400, WanleiBiotechnology Co., Ltd., Shenyang, China), NF-κB p65 polyclonal antibody (1:1,000, bs-0465R, Bioss, Beijing, China), p-NF-κB p65 polyclonal antibody (1:1,000, bs-0982R, Bioss, Beijing, China), and β-actin polyclonal antibody (1:5,000, bs-0061R, Bioss, Beijing, China) overnight at 4°C. Further, the membrane was incubated with antirabbit IgG peroxidases (bs-0061R, Bioss, Beijing, China) for 1 h at room temperature. The bands were visualized and imaged using ECL (Biosharp Life Sciences, Hefei, China) and gel imaging system (tanon-5200, Tianneng Technology Co., Ltd., Shanghai, China). Image J software was used for the analysis of gray values.
10
Immunohistochemical Analysis of Inflammatory Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The skin sections were deparaffinized, rehydrated and immunostained using conventional methods as below. The sections were incubated with 10% normal goat serum to reduce background staining caused by the secondary antibody. And then the sections were incubated with primary polyclonal antibody against IL-6 (1:200) (bs-0379R, Beijing Biosynthesis Biotechnology Co., Beijing, China), TNF-α (1:200) (bs-2150R, Beijing Biosynthesis Biotechnology Co.), NF-кB (1:200) (bs- 0465R, Beijing Biosynthesis Biotechnology Co.) for 12 h at 4°C. The control sections were treated with normal rabbit IgG rather than the primary antibody. The sections were then incubated with a secondary antibody, goat anti-rabbit lgG conjugated with biotin and peroxidase with avidin, using a rabbit ExtrAvidin staining kit (Sigma- Aldrich), followed by visualizing with 30 mg 3,3-diaminobenzidine (Wako, Tokyo, Japan) solution in 150 mL of 0.05 M Tris- HCl buffer, plus 30 μL H2 (link)O2 (link). Finally, the reacted sections for IL-6, TNF-α and NF- B were counterstained with hematoxylin solution (Merck, Tokyo, Japan). The specificity of the NF-кB antibodies in this amphibian was described by previous study.26 (link)
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!