For Western Blot (WB) analysis, frozen samples of renal cortex weighing 50–100 mg were lysed in RIPA buffer. An amount of 100 μg of total protein was then run on a 10% SDS-PAGE gel, as described elsewhere27 . Proteins were transferred onto a nitrocellulose membrane (Amersham Biosciences, Little Chalfont, UK) and the expression of the CASP1 protein was evaluated through the incubation of such membranes with a primary polyclonal rabbit anti-CASP1, antibody (#SC56036, Santa Cruz), using manufacturer recommended dilutions, followed by a peroxidase-conjugated antibody (Sigma). Bands were detected using an enhanced chemoluminescence system and analyzed with the Uvitec Cambridge® gel documentation device. Finally, membranes were stripped and probed with a primary anti-β-Actin antibody (Sigma) to confirm and estimate the loading and transfer proceedings. Further protein lysates obtained from frozen samples of renal cortex were used for ELISA analysis of the content of interleukin 1β (protein IL-1β) with a commercially available kit (#SRLB00, R&D Systems). This result was expressed as pg/ng of protein.
Srlb00
Manufactured by R&D Systems
Sourced in United States
The SRLB00 is a laboratory equipment product designed for general scientific and research applications. It is a versatile device that can be used for various tasks within a laboratory setting. The core function of this product is to provide a reliable and efficient platform for conducting experiments and analyses.
Automatically generated - may contain errors
Lab products found in correlation
Srta00, by R&D Systems (4 mentions)
Sr6000b, by R&D Systems (3 mentions)
Rta00, by R&D Systems (2 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Anti β actin antibody, by Merck Group (1 mentions)
Peroxidase conjugated antibody, by Merck Group (1 mentions)
Cambridge, by Uvitec (1 mentions)
Csb e04610r, by Cusabio (1 mentions)
Prta00, by R&D Systems (1 mentions)
Pr6000b, by R&D Systems (1 mentions)
Prlb00, by R&D Systems (1 mentions)
Quantikine elisa kit, by R&D Systems (1 mentions)
A001 1 for sod, by Nanjing Jiancheng (1 mentions)
A003 1 for mda, by Nanjing Jiancheng (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Naringin, by Merck Group (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
6 protocols using srlb00
1
Evaluating CASP1 and IL-1β in Renal Cortex
2
Cytokine Quantification in Arterial Specimens
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The ELISA method was used to determine the levels of TNF-α (cat. no. SRTA00; R&D Systems, Inc.), IL-1β (cat. no. SRLB00; R&D Systems, Inc.) and IL-18 (cat. no. CSB-E04610r; Cusabio Technology LLC) in the harvested artery specimens. Briefly, the harvested artery specimens were homogenized at 4°C, centrifuged at 500 × g and 4°C for 20 min, and then the supernatant was taken for ELISA detection following the manufacturer’s instructions.
3
Quantifying Inflammatory Mediators
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Quantikine® ELISA kits for IL-1β (RLB00, SRLB00, PRLB00), IL-6 (R6000B, SR6000B, PR6000B), and TNF-α (RTA00, SRTA00, PRTA00) were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). RvD1 (Cat# 500,380) was purchased from Cayman Chemical. (Ann Arbor, Michigan, USA). RvE1 (Cat# MBS2604861) and soluble receptor of AGE (sRAGE) (Cat# MBS722302) were purchased from MyBioSource, Inc. (San Diego, CA, USA), and LxA4 (Cat# 407,010) was purchased from Neogen Corporation (Lexington, Kentucky, USA). All measurements were performed according to the kit manufacturer’s instructions.
4
Neuroinflammation and Oxidative Stress
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STZ and naringin were purchased from Sigma Chemicals. Level of specific markers for oxidative stress including MDA and SOD in hippocampus tissues was analyzed by commercial kits (Cat. No: A003-1 for MDA, A001-1 for SOD, Nanjing Jiancheng Bioengineering Institute, China). The activities of TNF-α, IL-1β, and IL-6 were evaluated by the commercial immunoassay kits (Cat. No: RTA00 for TNF-α, SRLB00 for IL-1β, and R6000B for IL-6, R&D Systems, USA).
5
Inflammatory Markers in CKD Muscle
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The levels of inflammatory factors, such as serum CRP (R&D Systems, USA; DY1744), TNF-α (SRTA00), IL-1β (R&D Systems, USA; SRLB00), and IL-6 (R&D Systems, USA; SR6000B) in serum, were measured by colorimetric enzyme-linked immunosorbent assay (ELISA). And the levels of TNF-α, CRP, IL-1β, and IL-6 in gastrocnemius muscle in CKD rats were measured by qPCR. The primer sequences used are as follows: GAPDH: sense: 5-CTGGAGAAACCTGCCAAGTATG-3, antisense: 5-GGTGGAAGAATGGGAGTTGCT-3; IL-1β: sense: 5-TGATGAAAGACGGCACACCC-3, antisense: 5-TGTCCCGACCATTGCTGTTT-3; TNF-α: sense: 5-GCCACCACGCTCTTCTGTCTA-3, antisense: 5-CGCTTGGTGGTTTGCTACGA-3; IL-6: sense: 5-AAGCCAGAGTCATTCAGAGCAA-3, antisense: 5-GTCTTGGTCCTTAGCCACTCCT-3; and CRP: sense: 5-CCTTCGTATTTCCCGGAGTGTC-3, antisense: 5-CTCACATCAGCGTGGGCATAG-3.
Because macrophages play an important role in skeletal muscle repair and remodeling, the markers of M1 proinflammatory macrophages (CD68+/CD86+) and M2 anti-inflammatory macrophages (CD68+/CD206+) in muscle sections of different groups were also detected by performing immunofluorescence.
Because macrophages play an important role in skeletal muscle repair and remodeling, the markers of M1 proinflammatory macrophages (CD68+/CD86+) and M2 anti-inflammatory macrophages (CD68+/CD206+) in muscle sections of different groups were also detected by performing immunofluorescence.
6
Inflammatory Cytokine Analysis in Rats
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Once a week, approximately 1 mL of blood was collected from the tail vein in each rat. During blood collection, rats were anesthetized with 3% isoflurane. A twenty-one-gauge butterfly needle tip was inserted into a lateral tail vein, and the blood was collected into an anticoagulant EDTA-coated tube (Fisher Scientific; NH, United States). The tube was gently inverted several times to mix blood with the anticoagulant and transferred into an Eppendorf tube stored on ice until centrifugation at a speed of 3,000 rpm for 10 min. Blood plasma was then harvested and stored at −80°C for further processing. The levels of inflammatory cytokines in the blood plasma were analyzed by rat enzyme-linked immunosorbent assay (ELISA) for TNF-α (SRTA00, R&D Systems; MN, United States), IL-1β (SRLB00, R&D Systems), IL-6 (SR6000B, R&D Systems), and IFN-γ (SRIF00, R&D Systems) according to the manufacturer’s protocols. The plates were read on a spectrophotometric plate reader (Spectra Max M5, Molecular Devices; Sunnyvale, CA, United States). The cytokine levels before surgeries (W1) represented as baseline.
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