Proteinase inhibitor (PMSF, 1:100, Beyotime, Shanghai, PRC) was used to cleave fresh retina tissues and cells in RIPA lysis buffer. A total of 25 μg of protein was separated using SDS-PAGE (10%-12.5%), transferred onto PVDF membranes, and then incubated for an overnight period at 4 °C with the following primary antibodies: anti-SIRT1 (1:800, #27,523, Signalway Antibody), anti-NLRP3 (1:1000, ET1610-93, HUABIO), anti-IL-1β (1:800, WL02257, Wanleibio), and anti-Cleaved-IL-1β (1:800, WL00891, Wanleibio). On the following day, the membranes were with TBST, coated with BeyoECL Star (Beyotime, Shanghai, PRC), and incubated for one hour at room temperature with the secondary antibodies. GAPDH served as the internal control.
Wl00891
Manufactured by Wanlei
Sourced in China
The WL00891 is a laboratory equipment product. It serves as a core function related to the processing or analysis of samples in a laboratory setting.
Automatically generated - may contain errors
Lab products found in correlation
Wl00196, by Wanlei (2 mentions)
Wl03450, by Wanlei (2 mentions)
Il 18, by Wanlei (2 mentions)
Il 1β, by Wanlei (2 mentions)
Et1610 93, by Huabio (1 mentions)
Wl02257, by Wanlei (1 mentions)
Beyoecl star, by Beyotime (1 mentions)
Wla023, by Wanlei (1 mentions)
Ma5 23919, by Thermo Fisher Scientific (1 mentions)
Wl01372, by Wanlei (1 mentions)
Wl01980, by Wanlei (1 mentions)
Wla004, by Wanlei (1 mentions)
T1081, by Solarbio (1 mentions)
Wla003, by Wanlei (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Wl02635, by Wanlei (1 mentions)
Cbs 14787 1 ap, by Proteintech (1 mentions)
Wlp2412, by Wanlei (1 mentions)
Chemiscope 6100 system, by Clinx (1 mentions)
7 protocols using wl00891
1
Retinal Protein Expression Analysis
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
Western Blot Analysis of Protein Expression
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HUVECs were homogenized in RIPA lysis buffer and centrifuged at 20,000
g for 20 min at 4°C. Protein samples (10 μg) from independent experiments were electrophoresed on a 7.5%‒15% SDS-polyacrylamide gel and transferred onto PVDF membranes. Blots were blocked with 5% non-fat milk for 1 h at 37°C and incubated overnight at 4°C with antibodies against cystathionine gamma-lyase (CSE; sc-374249; Santa Cruz, Santa Cruz, USA), 3-mercaptopyruvate sulfotransferase (MPST; sc-374326; Santa Cruz), P21 (HA500005; HuaAn, Hangzhou, China), PPARδ (A5656; ABclonal, Shanghai, China), SGLT1 (A11976; ABclonal), SGLT2 (A20271; ABclonal), cystathionine beta-synthase (CBS; 14787-1-AP; Proteintech, Rosemont, USA), P53 (80077-1-RR; Proteintech), Caspase1 (WL03450; Wanlei, Shenyang, China), eNOS (WL01789; Wanlei), GLUT1 (WL01163; Wanlei), IL1β (WL00891; Wanlei), NLRP3 (WL02635; Wanlei), STAT3 (WL01836; Wanlei), and phosphorylated STAT3 (WLP2412; Wanlei). After being washed three times with TBST, the blots were incubated with anti-rabbit (SA00001-2; Proteintech) or anti-mouse IgG-HRP (SA00001-1; Proteintech). Antigen-antibody reactions were detected using ECL reagents (MERCK, Darmstadt, Germany) and detected with the ChemiScope 6100 system (CLiNX, Shanghai, China) for densitometric analysis.
g for 20 min at 4°C. Protein samples (10 μg) from independent experiments were electrophoresed on a 7.5%‒15% SDS-polyacrylamide gel and transferred onto PVDF membranes. Blots were blocked with 5% non-fat milk for 1 h at 37°C and incubated overnight at 4°C with antibodies against cystathionine gamma-lyase (CSE; sc-374249; Santa Cruz, Santa Cruz, USA), 3-mercaptopyruvate sulfotransferase (MPST; sc-374326; Santa Cruz), P21 (HA500005; HuaAn, Hangzhou, China), PPARδ (A5656; ABclonal, Shanghai, China), SGLT1 (A11976; ABclonal), SGLT2 (A20271; ABclonal), cystathionine beta-synthase (CBS; 14787-1-AP; Proteintech, Rosemont, USA), P53 (80077-1-RR; Proteintech), Caspase1 (WL03450; Wanlei, Shenyang, China), eNOS (WL01789; Wanlei), GLUT1 (WL01163; Wanlei), IL1β (WL00891; Wanlei), NLRP3 (WL02635; Wanlei), STAT3 (WL01836; Wanlei), and phosphorylated STAT3 (WLP2412; Wanlei). After being washed three times with TBST, the blots were incubated with anti-rabbit (SA00001-2; Proteintech) or anti-mouse IgG-HRP (SA00001-1; Proteintech). Antigen-antibody reactions were detected using ECL reagents (MERCK, Darmstadt, Germany) and detected with the ChemiScope 6100 system (CLiNX, Shanghai, China) for densitometric analysis.
4
Immunohistochemical Analysis of IL-1β and TLR4 in Jejunum
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Immunohistochemical staining was used to observe the protein expression and distribution in the jejunum following the steps described in earlier studies [10 (link)]. In brief, slices were deparaffinized twice in xylene and rehydrated in a graded series of ethanol. The antigen was repaired in sodium citrate buffer using a microwave oven and then cooled down at room temperature. For inactivation of endogenous peroxidase, 3% hydrogen peroxide (H2O2) was used, and tissues were incubated with 5% bovine serum albumin (BSA) (Boster, China) at 37°C for 30 min to block nonspecific binding sites. The primary antibody including rabbit anti-IL-1β (1:200) (WL00891, Wanleibio, China) and rabbit anti-TLR4 (1:500) (WL00196, Wanleibio, China) was used, and tissue sections were incubated at 4°C for 12 h. Then, horseradish peroxidase (HRP)-conjugated secondary antibody (Proteintech, China) was incubated for 30 min at 37°C. After DAB (Proteintech, China) staining, slices were counterstained with hematoxylin and mounted with coverslips.
5
Protein Expression Analysis in Heart Tissue
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Total protein from heart tissues was extracted using RIPA lysis buffer (Beyotime, Shanghai, China). A BCA protein assay kit (Beyotime, Shanghai, China) was used to measure the protein concentration. Protein samples (30 µg) were fractionated on 10% SDS‒PAGE gels and transferred onto PVDF membranes (GE Healthcare Life Sciences, Boston, United States ). The membranes were blocked with 5% skim milk for 1 h at room temperature and then incubated with the corresponding primary antibodies at 4°C overnight. The primary antibodies were as follows: Hif1a (Wanleibio, WL01607, 1:500), Ptgs2 (Proteintech, 27308-1-AP, 1:1,000), Gpx4 (Abcam, ab125066, 1:5,000), NRF2 (Abcam, ab92946, 1:1,000), Slc7a11 (Abcam, ab175186, 1:1,000), IL-1β (Wanleibio, WL00891, 1:500), TNF-α (Wanleibio, WL01581, 1:1,000) and GAPDH (Abcam, ab181602, 1:10,000). The membranes were incubated with an HRP-conjugated secondary antibody at room temperature for 1 h. Finally, the bands were detected by the FluorChem E System (ProteinSimple, United States ) using an enhanced chemiluminescence kit (Biosharp, Hefei, China). The protein intensity was determined by ImageJ software (NIH, United States ). GAPDH served as a loading control and all data were quantitatively normalized to the GAPDH.
6
Immunohistochemistry Analysis of Apoptosis Markers
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The tissue samples of the tumors, preserved in 2.5% glutaraldehyde-polyoxymethylene solution, were dehydrated and embedded in para n following routine methods. The para n sections were dewaxed and hydrated following routine methods. Rinsed the para n sections in PBS-T (3×5 min before each following steps), and then blocked with 3% peroxide-methanol at room temperature for endogenous peroxidase ablation. Afterwards, the sections were immersed in a boiling sodium citrate buffer for 15min, cooled down to room temperature. After that, incubated with blocking buffer (normal goat serum at room temperature for 20 min. Then incubated with primary antibody Caspase1(A nity, AF4005, 1:100), IL-1β (Wanleibio, WL00891, 1:100), IL-18 (Wanleibio, WL01127, 1:100) at 4℃ overnight. After that, the sections were incubated with the Secondary Goat anti-rabbit-IgG at 37℃ for 30min.Then incubated with the S-A/ HRP at 37°C for 30 min. Colored with 3,3-diaminobenzidin (DAB), and kept at room temperature without light for 10 min. After rinsing adequately with water, the sections were stained with hematoxylin for 5s, then dehydrated and sealed with neutral resins. We observed the sections under an upright microscope.
7
Immunohistochemistry Analysis of Apoptosis Markers
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The tissue samples of the tumors, preserved in 2.5% glutaraldehyde-polyoxymethylene solution, were dehydrated and embedded in para n following routine methods. The para n sections were dewaxed and hydrated following routine methods. Rinsed the para n sections in PBS-T (3×5 min before each following steps), and then blocked with 3% peroxide-methanol at room temperature for endogenous peroxidase ablation. Afterwards, the sections were immersed in a boiling sodium citrate buffer for 15min, cooled down to room temperature. After that, incubated with blocking buffer (normal goat serum at room temperature for 20 min. Then incubated with primary antibody Caspase1(A nity, AF4005, 1:100), IL-1β (Wanleibio, WL00891, 1:100), IL-18 (Wanleibio, WL01127, 1:100) at 4℃ overnight. After that, the sections were incubated with the Secondary Goat anti-rabbit-IgG at 37℃ for 30min.Then incubated with the S-A/ HRP at 37°C for 30 min. Colored with 3,3-diaminobenzidin (DAB), and kept at room temperature without light for 10 min. After rinsing adequately with water, the sections were stained with hematoxylin for 5s, then dehydrated and sealed with neutral resins. We observed the sections under an upright microscope.
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