Six rats in each group at different time points had brain tissues harvested for western blot analysis. Western blot was performed as previously described (Xu et al., 2017 (link)). Briefly, frozen perihematoma tissues (basal ganglia) were homogenized in RIPA lysis buffer (Beyotime, Shanghai, China). Then the protein samples were separated by 10% or 12% SDS-PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore). Then, the PVDF membranes were blocked with 5% bovine serum albumin for 1 h and incubated with the primary antibodies overnight, including: anti-sirt3 antibody (1:500, Abcam, ab86671), anti-NRF1 antibody (1:2000, Abcam, ab175932), anti-TFAM (1:1000, Abcam, ab131607), anti-SOD2 (1:5000, Abcam, ab13533), anti-Ac-SOD2 (1:1000, Abcam, ab137037), anti-cleaved caspase-3 (1:1000, CST, cst#9661), anti-Bax (1:1000, CST, cst#2772), anti-Bcl-2 (1:800, SantaCruz, sc-492), anti-NLRP3 (1:1000, ab210491, Abcam), anti-interleukin (IL)-1β (1:2000, Santa Cruz, sc-23459), and β-actin (1:5000, Abcam, ab8226). Then, the PVDF membranes were disposed with relevant secondary antibodies (1:5000) for 1 h at normal temperature. The signals of protein bands were detected with ChemiDoc detection system and quantified using Quantity One software (Bio-Rad, Hercules, CA, United States).
Ab86671
Manufactured by Abcam
Sourced in United States
Ab86671 is a lab reagent product offered by Abcam. It is designed for use in various laboratory applications, but no further details about its core function or intended use can be provided in an unbiased and factual manner without risk of interpretation or extrapolation.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sc 492, by Abcam (1 mentions)
Ab175932, by Abcam (1 mentions)
Ab131607, by Abcam (1 mentions)
Ab13533, by Abcam (1 mentions)
Ab210491, by Abcam (1 mentions)
Ab8226, by Abcam (1 mentions)
Chemidoc detection system, by Bio-Rad (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ap0060, by Bioworld Technology (1 mentions)
Ab54481, by Abcam (1 mentions)
Dh351 2, by Dingguo (1 mentions)
Dapi c1002, by Beyotime (1 mentions)
Ecl method, by Merck Group (1 mentions)
Ab68155, by Abcam (1 mentions)
Ab109199, by Abcam (1 mentions)
Ab51243, by Abcam (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
8 protocols using ab86671
1
Western Blot Analysis of Brain Tissues
2
Quantification of PGC-1α and SIRT3 in Neurons
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The samples from primary neurons in vitro and cerebral cortices in vivo were lysed with RIPA buffer (Beyotime, Jiangsu, China). After protein concentrations were measured, the same amount of protein from every sample was separated and transferred. After blocking with defatted milk, the membranes were hatched with primary antibodies overnight at 4°C against PGC-1α (1: 2000, ab54481; Abcam, Cambridge, MA, USA), SIRT3 (1: 1000, ab86671; Abcam), and β-actin (1: 5000, AP0060; Bioworld Technology, Minneapolis, MN, USA). After washing with PBS containing Tween-20 (PBST), the membranes were incubated with appropriate secondary antibodies at room temperature. After incubation of the chemiluminescence solution, the protein bands were visualized. Band density was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA).
3
Immunofluorescence Analysis of Sirt3 Expression
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Cells were fixed in 4% paraformaldehyde (8009628, Sinopharm Chemical Reagent) or 15 min, permeabilized in 0.5% Triton X‐100 (DH351‐2, Beijing Dingguo Changsheng Biotechnology) in PBS for 5 min, blocked with 4% goat serum in PBS for 60 min, and incubated with rabbit monoclonal anti‐Sirt3 (Ab86671, Abcam) overnight at 4°C. DyLight 488‐conjugated goat anti‐rabbit (GAR4882, Multi Sciences) was used as a secondary antibody. Samples were stained with DAPI (C1002, Beyotime) before photographed on a fluorescence microscope (FV1000MPE‐share).
4
Protein Expression Analysis of Lung Tissue
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Frozen right lung tissues were homogenized with RIPA buffer containing protease inhibitor. Aliquots of tissue lysates were used for protein analysis and then separated on 12% SDS–PAGE. Separate proteins were transferred onto the Immobilon-P transfer membranes 0.45 mm (Millipore). The membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.05% Tween 20 (TBST). After blocking for 3 h, the membrane was incubated with primary antibodies (1:500 dilutions) in blocking buffer for 24h at 4°C. Primary antibodies against SOD2 (ab68155), SIRT3 (ab86671), p16 (ab51243) and p21 (ab109199) were purchased from Abcam. After washing with TBST for 3 times (10 min each), the membranes were incubated with secondary anti-rabbit antibody (1:10,000 dilutions in 5% skim milk) linked to HRP for 1 h at room temperature, and then the membranes were developed using ECL method (Millipore, Billerica, U.S.A). β-actin was used as a loading control. ImageJ densitometry software (Version 1.41; National Institutes of Health, Bethesda, MD) was used for the band quantification of Western blots.
5
Immunofluorescent Analysis of SIRT1 and SIRT3
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Firstly, samples were deparaffinized in xylene, rehydrated in a series of graded alcohol, and subjected to antigen retrieval. Secondly, sections were blocked with 5% bovine serum albumin (BSA) in PBS, exposed to 0.5% Triton X-100 for 2 h, and then incubated with anti-SIRT1 (mouse monoclonal, ab110304, Abcam, UK, 1 : 200) and anti-SIRT3 (rabbit polyclonal, ab86671, Abcam, UK, 1 : 200) overnight at 4°C. The sections were washed with PBS three times and incubated with secondary antibody (Alexa Fluor 488, bs-0295G-AF488, Bioss, China, 1 : 200; excitation spectrum 495 nm, emission spectrum 519 nm, cy3, bs-0368R-Cy3, Bioss, China, 1 : 200; excitation spectrum 552, emission spectrum 570) for 2 h at 37°C. Images were captured under laser confocal microscope (FV1000, Olympus Optical Co., Ltd., Japan). The average optical density of positive expression was quantified by the Image-Pro Plus 6.0 software (Media Cybernetics, USA).
6
Immunohistochemical and Histological Analysis of Intracerebral Hemorrhage in Rats
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Rats were sacrificed under deep pentobarbital anesthesia at 24 h after ICH, and then intracardially perfused with 0.1 mmol PBS and 4% paraformaldehyde (PFA). Coronal cryosections were preprocessed with 10% donkey serum and 0.3% triton X-100. Then the brain cryosections were incubated at 4°C with anti-sirt3 antibody (1:200, Abcam, ab86671), cleaved caspase-3 antibody (1:250, Cell Signaling Technology, CST#9661), Iba-1 (1:500, Abcam ab5076), NeuN (1:500, Abcam, ab177487). Twelve hours later, cryosections were incubated with secondary antibody [Thermo Fisher Scientific, Donkey anti-Rabbit IgG (H+L) Alexa Fluor 488, Donkey anti-Mouse IgG (H+L) Alexa Fluor 594, Goat anti-Rabbit IgG (H+L) Alexa Fluor 488]. Then, the fluorescence microscope (Olympus, Tokyo, Japan) was used to capture the images.
As described previously (Xu et al., 2006 (link)) brain samples were dissected and embedded in paraffin. Five to eight micrometers of coronal sections were stained with 0.1% cresyl violet hematoxylin and eosin (H&E) and prepared for the subsequent microscope.
As described previously (Xu et al., 2006 (link)) brain samples were dissected and embedded in paraffin. Five to eight micrometers of coronal sections were stained with 0.1% cresyl violet hematoxylin and eosin (H&E) and prepared for the subsequent microscope.
7
Immunofluorescence Staining of SIRT3
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Isolated ECs from WT mice were washed with PBS and fixed in 4% formaldehyde for 10 minutes at room temperature and then incubated with 5% normal goat serum in 0.3% Triton X‐100/PBS for 1 hour to permeabilize the cells and block nonspecific protein–protein interaction. The cells were then incubated with the rabbit anti‐SIRT3 antibody (Abcam, ab 86671, 1:100) in 1% BSA in 0.3% Triton X‐100/PBS overnight at +4°C, followed by secondary Cy™3 donkey anti‐rabbit antibody (1:200; No. 711‐165‐152; Jackson Immunoresearch Laboratories, West Grove, PA) for 1 hour; 4′,6‐diamidino‐2‐phenylindole was used to stain the cell nuclei.
8
Protein Expression Analysis of HEI-OC1 Cells
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The cultured HEI-OC1 cells were washed three times with phosphate-buffered saline (PBS) and placed on ice with cold RIPA buffer (Millipore, Billerica, MA, United States) for 30 min. The protein content was determined using a bicinchoninic acid protein assay kit (Beyotime, Shanghai, China). The protein was transferred onto a polyvinylidene fluoride membrane for SDS-PAGE. The membrane was sealed with 5% bovine serum albumin for 2 h and incubated overnight at 4°C with primary antibodies. After washing three times with Tris-buffered saline containing Tween 20, the membrane was incubated with the secondary antibody (1:10,000; Absin) at room temperature (24°C) for 2 h. An ECL Kit (Affinity Biosciences, Cincinnati, OH, United States) was used to visualize the protein signals. The following antibodies were used: Rabbit anti-Bcl-2 (No. ab182858 1:1,000, Abcam, United States), rabbit anti-Bax (No. ab32503, 1:1,000, Abcam), rabbit anti-caspase-3 (No. ab13847, 1:1,000, Abcam), rabbit anti-SIRT3 (No. ab86671, 1:1,000, Abcam), rabbit anti-PGC-1α (No. AF5395, 1:1,000, Affinity), rabbit anti-β-Tubulin (No. ab18207, 1:1,000, Abcam), rabbit anti-p-AMPK (No. ab133448, 1:3,000, Abcam), and AMPK (No. ab32047, 1:3,000, Abcam). First photograph of Western Blot can be found in Supplementary Figures 4-6 .
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