Ovarian tissues and GCs were homogenized in RIPA lysis buffer (P0013B, Beyotime) containing 1 mM Pierce™ Phosphatase Inhibitor (B15001, Selleck, Texas, USA) and 0.1% Halt™ Protease Inhibitor Cocktail (B14001, Selleck). The gel electrophoresis system of Bio-Rad (12% SDS polyacrylamide) was used to separate proteins from samples that contained an identical quantity of protein (30 μg); the proteins were then transferred onto polyvinylidenedifluoride membranes (IPVH00010, Merck Millipore, Massachusetts, USA). Target bands were blocked with 5% BSA for 2 h at 24°C. Subsequently, they were incubated with primary antibodies against GRP-78 (1 : 1000), CHOP (1 : 1000), PERK (1 : 1000, 20582-1-AP, Proteintech, Chicago, USA), ATF-6 (1 : 1000, 24169-1-AP), IRE1α (1 : 1000), p-IRE1α (1 : 1000), XBP1 (1 : 500, WL00708, Wanleibio, Shenyang, China), Bax (1 : 5000, 50599-2-Ig, Proteintech), caspase-9 (1 : 1000, 10380-1-AP), cleaved-caspase-3 (1 : 1000, 66470-2-Ig), Ar (1 : 1000), Cyp19α1 (1 : 1000), Cyp11α1 (1 : 1000), and GAPDH (1 : 5000, MB9231, Bioworld) overnight at 4°C, followed by the addition of HRP-labeled secondary antibodies. Blots were visualized using chemiluminescent detection. Densitometric analysis was performed using Image J.
Wl00708
Manufactured by Wanlei
Sourced in China, United Kingdom
The WL00708 is a general-purpose laboratory equipment designed for a variety of applications. It functions as a tool to perform essential tasks in research and analysis settings.
Automatically generated - may contain errors
Lab products found in correlation
Gel electrophoresis system, by Bio-Rad (1 mentions)
Ipvh00010, by Merck Group (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Ab124945, by Abcam (1 mentions)
Ab33889, by Abcam (1 mentions)
Af3242, by Affinity Biosciences (1 mentions)
C1002, by Beyotime (1 mentions)
Wl02400, by Wanlei (1 mentions)
Loading buffer 5, by Beyotime (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (1 mentions)
3 protocols using wl00708
1
Ovarian Granulosa Cells Protein Analysis
2
Ovarian Tissue Immunohistochemistry and Immunofluorescence
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To evaluate protein localization and intensity, immunohistochemistry and immunofluorescence were performed according to previously described methods [26 (link), 27 (link)]. Ovarian tissue slides were dewaxed in xylene and graded series of ethanol solutions. Slides were then fixed in 4% paraformaldehyde and blocked with 3% bovine serum albumin (BSA) at 37°C. The sections were incubated overnight at 4°C with antibodies against p-IRE1α (1: 200, ab124945, Abcam, Cambridge, UK), XBP1 (1: 100, WL00708, Wanleibio, Shenyang, China), p-PI3K (1 : 200, AF3242, Affinity, USA), p-AKT (1: 200, 4060T, CST, USA), p-p53 (1: 200, ab33889, Abcam, UK), p-NF-κB (1: 200, 3033S, CST, USA), and cleaved-caspase-3 (1: 200, 66470-2-Ig, Proteintech, USA). All secondary antibodies were diluted (1 : 2000) and incubated at 25°C for 2 h. The sections were processed according to the avidin-biotinylated-peroxidase complex and DAB staining techniques, followed by observation under an optical microscope. Nuclei were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (1 : 2000, C1002, Beyotime, China) for 30 min and photographed using an Olympus laser scanning confocal microscope (FV3000).
3
Protein Expression Analysis of Spinal Cords
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The isolated spinal cords were homogenated with RIPA buffer (Beyotime Biotechnology, Shanghai, China) containing 1% protease inhibitor (Beyotime Biotechnology, Shanghai, China) for 30 minutes on ice. The homogenates were centrifuged at 12000 g for 25 minutes at 4°C and supernatants were collected. NanoDrop 2000C (Thermo Scienti c, USA) was then used for protein concentrations determination. The homogenates and loading buffer 5× (Beyotime Biotechnology, Shanghai, China) were mixed at a ratio of 4: 1, denatured in the metal bath (100°C, 8~10 mins), cooled to room temperature and loaded. Protein samples were subjected to 12.5% SDS-PAGE (Epizyme Biotech, Shanghai, China) and then transferred to polyvinylidene uoride (PVDF) membranes. Membranes were blocked in protein free rapid blocking buffer (Epizyme Biotech, Shanghai, China) for 15 minutes at room temperature. After blocking, membranes were incubated with the primary antibodies against ATF3 (DF6660, A nity Biosciences), XBP1 (WL00708, WanleiBio), HMOX1 (WL02400, WanleiBio), DDIT3 (GADD153, Proteintech), CHAC1 (DF9353, A nity Biosciences) and GAPDH (AF7021, A nity Biosciences) overnight at 4°C. Membranes were incubated with the secondary antibody at room temperature for 1.5 h. Protein bands were captured using an ECL chemiluminescence system (Epizyme Biotech, Shanghai, China).
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