Cells from blood samples were treated with ammonium chloride-based buffer (10X RCB lysis buffer) followed by a series of washes with a phosphate buffer solution (PBS) of pH 7.4. Cells were suspended at a concentration of 1x 106 cells/ml and incubated with a rabbit anti-SIRT1 antibody (Abcam) for 20 min at 4°C, followed by incubation with an anti-rabbit FITC secondary antibody (eBioscience) for 30 min to detect SIRT1-positive cells. After washing with PBS, the cells were resuspended in a solution with 1% p-formaldehyde and stored at 4°C until analysis in the FACSCanto II flow cytometer (BD). The results were expressed as the percentage of positive cells.
Rabbit anti sirt1 antibody
Manufactured by Abcam
Sourced in United States
Rabbit anti-SIRT1 antibody is a primary antibody that specifically recognizes the SIRT1 protein. SIRT1 is a NAD-dependent deacetylase that plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Facscanto 2, by BD (1 mentions)
Anti β actin antibody, by R&D Systems (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Rabbit anti p21, by Cell Signaling Technology (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Vectashield, by Vector Laboratories (1 mentions)
Rabbit anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
Ecl detection kit, by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Rabbit anti bcl 2 antibody, by Abcam (1 mentions)
Hrp labelled goat anti mouse igg h l, by Beyotime (1 mentions)
Mouse anti gapdh antibody, by Proteintech (1 mentions)
Hrp labelled goat anti rabbit igg h l, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
7 protocols using rabbit anti sirt1 antibody
1
SIRT1 Expression in Blood Cells
2
Protein Quantification and Immunoblotting Protocol
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For protein quantification cells were washed with PBS, resuspended in lysis buffer (HEPES 100 mmol/L, 1 mol/L NaCl, 200 mmol/L MgCl2, 100 mmol/L EDTA and Triton X-100, pH 7.4) and lysed by sonication at 50% amplitude. Then, the cells were centrifuged at 12,000 rpm (2400 × g) at 4 °C for 30 min. The supernatant was used to determine protein concentrations by the Bradford method (BCA Protein Assay, Thermo Scientific; Rockford, IL, USA). For immunoblots, 50 μg of total protein of HeLa, 3T3 and PBMC samples were electrophoresed through 4%–15% SDS-polyacrylamide gel, transferred to 0.45 μm nitrocellulose membranes (Millipore, Billerica, MA, USA) on a semi-dry electro transferring unit (Trans-Blot Turbo Transfer System, Bio-Rad Laboratories) and immunoblotted using a rabbit polyclonal anti-NAT1 antibody (1: 1000), mouse polyclonal anti-NAT2 antibody (1: 500), rabbit anti-SIRT1 antibody (1:500; Abcam) or mouse monoclonal anti-SIRT6 antibody (1: 3000), and then incubated with HRP-conjugated secondary antibody for 1 h. Finally, anti-β-actin antibody (R&D) was used as a loading and transfer control. The membranes were revealed by chemiluminescence. The intensities of the bands were detected using the ChemiDoc™ XRS+ System (Bio-Rad).
3
Molecular Mechanisms of Capsaicin-Induced Cell Signaling
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In this study, the following antibodies were used. Rabbit anti-p21, phospho-p38 and GAPDH antibodies were purchased from Cell Signaling (Danvers, MA, USA). Rabbit anti- Sirt1 antibody was from Abcam (Cambridge, MA, USA). Recombinant capsaicin was purchased from Abcam (Cambridge, MA, USA).
4
Immunohistochemical Quantification of SIRT1 in Liver
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Liver specimens fixed in 10% buffered formalin were embedded in paraffin blocks. Sections (5 μm thick) of the liver were processed using a standard immunostaining protocol. After routine deparaffinization, hydration, and blockage of endogenous peroxidase, sections were pretreated by microwave for 20 min in 10 mmol/L sodium citrate buffer (pH 6.0) for antigen retrieval and followed by incubation sequentially with blocking agent, rabbit anti-SIRT1 antibody (1 : 100, Abcam, Cambridge, MA, USA) and secondary antibody (1 : 200, Promega, Madison, WI, USA).
Slides were counterstained with hematoxylin after 3 min of diaminobenzidine reaction, and cover slipped using Vectashield (Vector Labs, Burlingame, CA, USA), then photographed and converted to a digital image using light microscopy equipped with a camera (Olympus CX31, NY, USA). Negative control was carried out by omitting the primary antibody. Positive staining (dark brown) for SIRT1 in livers was quantified by two investigators in a blinded manner at a magnification of 400x using image analysis software Image-Pro Plus, and the results were expressed as the ratio of the mean of normal SD rats. Three slides were chosen at random in each animal for quantification (total 24 slides for each group).
Slides were counterstained with hematoxylin after 3 min of diaminobenzidine reaction, and cover slipped using Vectashield (Vector Labs, Burlingame, CA, USA), then photographed and converted to a digital image using light microscopy equipped with a camera (Olympus CX31, NY, USA). Negative control was carried out by omitting the primary antibody. Positive staining (dark brown) for SIRT1 in livers was quantified by two investigators in a blinded manner at a magnification of 400x using image analysis software Image-Pro Plus, and the results were expressed as the ratio of the mean of normal SD rats. Three slides were chosen at random in each animal for quantification (total 24 slides for each group).
5
Western Blot Analysis of MMP-13, SIRT1, and NF-κB
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The cells were washed twice with PBS and then lysed, homogenized, sonicated, and centrifuged. The protein concentration in the supernatant was determined by using a protein assay reagent (Bio-Rad, Hercules, CA). Samples and prestained molecular weight markers were separated by SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA). The membranes were blocked in 5% skim milk in PBS containing 0.05% Tween-20 (PBS-Tween) for 2 h. The membranes were incubated in PBS-Tween containing rabbit anti-MMP-13 antibody (Abcam, Cambridge, MA; diluted at 1:400), rabbit anti-SIRT1 antibody (Abcam; diluted at 1:1,000), rabbit anti-acetyl-p65 antibody (Abcam; diluted at 1:500), or rabbit anti-β-actin antibody (Santa Cruz Biotechnology, Santa Cruz, CA; diluted at 1:1,000) for 1 h at ambient temperature and then incubated overnight at 4°C. The following day, it was incubated for another 2 h at ambient temperature with anti-rabbit secondary horseradish peroxidase-linked antibody (CST, Danvers, MA; diluted at 1:2,000). The peroxidase activity on the PVDF membrane was visualized on X-ray films by using an ECL detection kit (Amersham Pharmacia Biotech, Chalfont St Giles, UK), according to the manufacturer’s protocol.
6
Western Blot Analysis of Skin Fibroblast Proteins
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Skin primary fibroblasts were harvested when cells were at a confluency of 80%‐90%. The protein content was measured with BCA protein assay kit (Beyotime). Denatured proteins (20 μg) were separated by 10% SDS‐PAGE and transferred to 0.45 μm PVDF membrane, blocked with TBST containing 5% skim milk or 5% BSA and incubated with rabbit anti‐SIRT1 antibody (1:1000, Abcam), rabbit anti‐Smad2/3 antibody (1:1000, CST), rabbit anti‐Smad4 antibody (1:1000, CST), rabbit anti‐YAP/TAZ antibody (1:1000, CST), rabbit anti‐MMP1 antibody (1:1000, ProteinTech Group), rabbit anti‐COL1 antibody (1:1000, Abcam), rabbit anti‐BCL2 antibody (1:1000, Abcam) and mouse anti‐GAPDH antibody (1:10 000, ProteinTech Group) at 4°C overnight. The membranes were incubated with HRP‐labelled goat anti‐rabbit IgG (H + L) (1:1000, Beyotime) or HRP‐labelled goat anti‐mouse IgG (H + L) (1:1000, Beyotime) for 1.5 hours at room temperature. The blots were visualized with enhanced ECL (Beyotime) following exposure to DNR MF‐ChemiBIS 2.0 (ISR). All experiments were tested at least three times.
7
Evaluation of EMPA and HAL Effects
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EMPA was obtained from Boehringer Ingelheim (Ingelheim, Germany). HAL was obtained from Kahira Pharmaceuticals (Cairo, Egypt). Anti-mullerian hormone (AMH) (Catalog No: MBS701712), follicle-stimulating hormone (FSH) (Catalog No: MBS2502190), luteinizing hormone (LH) (Catalog No: MBS764675), Nrf2 (Catalog No: MBS752046) and caspase-3 (Catalog No: MBS261814) were measured by ELISA kits (MyBioSource, San Diego, USA). TNF-α (Catalog No: BMS622), IL-6 (Catalog No: ERA31RB) ELISA kits were obtained from Thermo Fisher Scientific Inc., Waltham, MA, USA). Rabbit anti-beta-actin antibody (1:1000, Catalog No: ab8227, Abcam, Cambridge, UK) and Rabbit anti-Sirt1 antibody (1:1000, Catalog No: ab189494, Abcam) were applied.
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