Brain tissue with the range of +2.76 mm to −3.24 mm/Bregma was stained from each brain, and 3 random nonoverlapping fields were assessed from each section by a blinded observer using Image-Pro Plus 6.0 software. For immunohistochemical analysis for p-Akt, p-mTOR of the brain tissue was performed on representative serial paraffin-embedded sections as previously described. They were stained with rabbit polyclonal phospho-Akt antibody (CST, USA, 1 : 100) or rabbit polyclonal phospho-mTOR antibody (CST, USA, use a concentration of 5 μg/ml) followed by anti-antibody of HRP-conjugated goat anti-rabbit IgG H&L (1 : 7500 in TBS-T, Abcam, UK). Standard fluorescent immunohistochemistry procedures and the primary and secondary antibodies indicated below were used to treat the brain tissue for immunofluorescence: rabbit polyclonal anti-LC3 antibodies (Abcam, UK, use a concentration of 1 μg/ml) and HRP-conjugated goat anti-rabbit IgG H&L (1 : 7500 in TBS-T, Abcam, USA). PC12 cells were fixed by formaldehyde and blocked by Donkey serum and then were processed with standard immunofluorescent techniques using primary and secondary antibodies of goat polyclonal to NLRP3 (Abcam, UK, use a concentration of 10 μg/ml), rabbit monoclonal to NF-κB p65 (Abcam, UK, 1 : 100), and secondary antibody. The nuclear area was defined according to DAPI staining.
Tbs t
Manufactured by Abcam
Sourced in United Kingdom, United States
TBS-T is a buffer solution commonly used in various laboratory procedures. It is a mixture of Tris-buffered saline (TBS) and the detergent Tween-20. TBS-T is primarily used as a washing buffer to remove unbound materials during immunoassays, Western blotting, and other similar techniques.
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Lab products found in correlation
Hrp conjugated goat anti rabbit igg h l, by Abcam (1 mentions)
Electrochemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Fusion fx5, by Vilber (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Bicinchoninic acid (bca), by Beyotime (1 mentions)
Antigen retrieval solution, by Agilent Technologies (1 mentions)
Tbs t, by Merck Group (1 mentions)
Z fix, by Anatech (1 mentions)
Hrp conjugated goat anti rabbit igg secondary antibody, by Abcam (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Radioimmunoprecipitation assay (ripa), by Abcam (1 mentions)
Horseradish peroxidase hrp conjugated anti rabbit igg antibody, by Merck Group (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Laemmli sample buffer, by Bio-Rad (1 mentions)
Tween 20, by Bio-Rad (1 mentions)
Gradient tgx gels, by Bio-Rad (1 mentions)
Ab216130, by Abcam (1 mentions)
Ab41927, by Abcam (1 mentions)
Hrp conjugated anti rabbit igg secondary antibody, by Bio-Rad (1 mentions)
Trans blot sd semi dry transfer cell, by Bio-Rad (1 mentions)
10 protocols using tbs t
1
Immunohistochemical Analysis of Brain Tissue
2
Quantification of HIF-1α and Endostatin Proteins
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Tissues were lysed using radioimmunoprecipitation assay buffer (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany). The soluble protein concentration was determined by the Bradford method. Total protein (10 µg) was resolved on 10–15% SDS-PAGE and electro-transferred onto polyvinylidene difluoride membranes. The membranes were blocked with PBS containing 1% Tween 20 at 4°C overnight, washed and incubated overnight at 4°C with anti-HIF-1α (#AH339-2) and anti-endostatin (#BAF1098) primary antibodies (1:1,000 dilution in TBS-T; Bioworld Technology, Inc., St. Louis Park, MN, USA). Upon being washed, membranes were incubated for 1 h at 22–25°C with the corresponding secondary antibodies (1:1,200 dilution in TBS-T; #A00131-1; Abcam) for 1 h at room temperature. The immunoblots were detected using an electrochemiluminescence kit (Pierce Biotechnology, Inc., Rockford, IL, USA) and exposed to the Fusion FX5 automatic gel imaging analysis system (Vilber Lourmat, Marne-La-Vallée, France).
3
Protein Extraction and Western Blot
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Total protein from approximately 1×106 cells were extracted using radioimmunoprecipitation assay buffer and then quantified using the bicinchoninic acid method (Beyotime, Shanghai, China). Equal amounts of protein sample were separated using 8–15% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat milk in TBST (Abcam, Cambridge, UK) for 1 h at room temperature and incubated with primary antibody (Abcam) at 4°C overnight. Membranes were then washed three times andincubated with horseradish peroxidase-labeled secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h. Signals were detected using an enhanced Chemiluminescent Western Blot Analysis Kit (Thermo Fisher Scientific, Inc.).
4
Histological Analysis of Angiogenesis
5
Western Blot Analysis of Stra8 and GFRA1
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Proteins were extracted from testicular tissue specimens using radioimmunoprecipitation assay buffer (RIPA, Abcam, Germany) supplemented with protease and phosphatase inhibitors and centrifuged. After determining the concentration of proteins using BCA Protein Assay Kit (Abcam, USA), an equal amount of the extracted protein in each sample was separated using SDS-PAGE and transferred to the nitrocellulose membrane. Following blockage of membranes in 5% non‐fat dry milk for 2 h at room temperature, the membranes were incubated with primary antibodies against Stra8 (1:1000, Abcam, USA) and Gfra1 (1:1000, MyBioSource, Inc.) at 4 °C overnight. Then, the membranes were washed three times with Tris buffered saline with 0.1%Tween 20 (TBS-T, Abcam, Germany) and incubated with horse radish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:2000, Abcam, USA) for 1 h at room temperature. The expression rate of Stra8 and GFRA1 proteins was assessed using enhanced chemiluminescence. Finally, the intensity of the bands was normalized to GAPDH and analyzed using the ImageJ software.
6
Osteogenic Differentiation Assay with TSG Treatment
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The cells were inoculated in 6-well plates and cultured for 24 h for osteogenesis induction. The treatment group was given TSG at concentrations of 1, 10, and 50 μmol/L. The blank induction medium was added to the control group, and the culture medium in both the administration group and the control group contained 1‰ DMSO. After 3 days, the total protein was extracted from each well with 300 μL cell lysate (containing PMSF 1 mmol/L). The protein concentration was detected by BCA method. Sample loading buffer containing bromophenol blue was added and denatured at 95°C for 5 min. After 12% SDS-PAGE electrophoresis, the protein was transferred to a PVDF membrane. The shaker was sealed with 5% skimmed milk powder at room temperature for 1 h. KDM5A primary antibody diluted with TBST (Abcam, dilution ratio: 1 : 1000) was added and left at 4°C overnight. The next day, the PVDF membrane was washed three times with TBST, and then horseradish peroxidase-labeled secondary antibody diluted with TBST was added. The cells were shaken at room temperature for 2 h. After washing the film 3 times, the gray value of the protein was detected by an exposure meter.
7
Western Blot Analysis of Transfected Cells
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Transfected or infected cells were recovered by scraping, washed twice in PBS and lysed for 30 min on ice with Tris-NaCl lysis buffer (0.01 M Tris–HCl, 0.1 M NaCl, 2 mM PMSF, and 1% NP-40), 48 hr post transfection. The lysates were centrifuged (800 g), and the protein was quantitated by Bradford assay and was separated with SDS page. Samples were prepared by heating to 90 °C in SDS sample buffer for 5 min. Proteins were resolved by 12% SDS-PAGE and transferred to nitrocellulose. The membrane was blocked in 3% non-fat skimmed milk in TBS plus 0.05% Tween 20 (TBS-T) for 2 hr at room temperature (Abcam, UK). To detect the antigen-antibody complex, the membrane was washed and then incubated with Horseradish Peroxidase (HRP) conjugated anti-rabbit IgG antibody diluted 1:10,000 (Sigma, USA) for 1 hr at room temperature, washed again and followed by developing with diaminobenzidine (Roche, Germany).
8
Extracellular Vesicle Marker Analysis
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Extracellular vesicles were assessed for the EV-specific markers CD63 and Flotillin-1 (Flot-1), using western blotting. EV samples were diluted 1:1 in denaturing 2×Laemmli sample buffer (containing 5% beta-mercaptoethanol, BioRad, United Kingdom) and heated for 5min at 100°C. Protein separation was carried out at 165V using 4–20% gradient TGX gels (BioRad United Kingdom), followed by western blotting at 15V for 1h using a Trans-Blot® SD semi-dry transfer cell (BioRad, United Kingdom). Membranes were blocked with 5% bovine serum albumin (BSA, Sigma, United Kingdom) in Tris buffered saline (TBS) containing 0.1% Tween20 (BioRad, United Kingdom; TBS-T) for 1h at RT and primary antibody incubation was carried out overnight at 4°C using the EV-marker CD63 (ab216130, Abcam, United Kingdom) and Flot-1 (ab41927, Abcam); diluted 1/1,000 in TBS-T. The membranes were then washed at RT in TBS-T for 3×10min and thereafter incubated with HRP-conjugated anti-rabbit IgG secondary antibodies (BioRad), diluted 1/3,000 in TBS-T, for 1h at RT. The membranes were then washed for 4×10min TBS-T, and visualised, using enhanced chemiluminescence (ECL, Amersham, United Kingdom) in conjunction with the UVP BioDoc-ITTM System (Thermo Fisher Scientific, United Kingdom).
9
Purification and Characterization of rAlt a 1
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The supernatant was filtered through a 0.22-μm membrane (Millipore, Burlington, MA, USA) and applied to a Ni-IDA resin that was washed and equilibrated with 20 column volumes in a binding buffer at a flow rate of 1.5 ml/min. Ni-IDA elution buffer (20 mM Tris, 0.15 M NaCl, 500 mM imidazole) was then passed through the column with linear imidazole concentrations (0–50%) for 30 min. rAlt a 1 protein was eluted at a 200 mM imidazole concentration.
Purity was assessed by 12% SDS-PAGE (3 μg/slot) under reducing and non-reducing conditions. The concentration of rAlt a 1 was 1.65 mg/ml, as detected using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For immunoblotting studies, the separated proteins were transferred to polyvinylidene fluoride (PVDF, Millipore, Burlington, MA, USA) membranes and blocked with 5% non-fat milk in TBST (Solarbio, Beijing, China) for 2 h at room temperature, followed by incubation at 4°C overnight with human sera (1:20) and anti-His tag antibody (Sangon Biotech, Shanghai, China). After washing with TBST for four times, the blots were incubated with horseradish peroxidase (HRP)-conjugated anti-human IgE (Abcam, Cambridge, MA, USA) and goat anti-mouse IgG antibody (Sangon Biotech, Shanghai, China) for 1 h. Finally, the blots were detected by enzyme-linked chemiluminescence.
Purity was assessed by 12% SDS-PAGE (3 μg/slot) under reducing and non-reducing conditions. The concentration of rAlt a 1 was 1.65 mg/ml, as detected using the BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). For immunoblotting studies, the separated proteins were transferred to polyvinylidene fluoride (PVDF, Millipore, Burlington, MA, USA) membranes and blocked with 5% non-fat milk in TBST (Solarbio, Beijing, China) for 2 h at room temperature, followed by incubation at 4°C overnight with human sera (1:20) and anti-His tag antibody (Sangon Biotech, Shanghai, China). After washing with TBST for four times, the blots were incubated with horseradish peroxidase (HRP)-conjugated anti-human IgE (Abcam, Cambridge, MA, USA) and goat anti-mouse IgG antibody (Sangon Biotech, Shanghai, China) for 1 h. Finally, the blots were detected by enzyme-linked chemiluminescence.
10
Western Blot Analysis of EMT Markers
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The cell proteins were extracted using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA), after which the protein concentration was determined by a bicinchoninic acid protein assay kit (BCA, Sigma-Aldrich; Merck KGaA). Protein samples (40 μg/lane) were separated by 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with Tris-buffered saline and 0.1% Tween 20 (TBS-T) containing 5% bovine serum albumin, then incubated with primary antibody against E-cadherin, vimentin, and TWIST1 (diluted 1:1000 in TBS-T; ABCAm, Cambridge, MA, USA). The membranes were incubated at 4°C overnight. After washing with TBS-T three times, the membranes were incubated with the appropriate horseradish peroxidase–labeled secondary antibody (diluted 1:2000 in TBS-T; ABCAm) at 37°C for 2 h. GAPDH was used as the internal control. The proteins were examined using enhanced chemiluminescence reagents (Pierce, Rockford, IL, USA) and the band density was scanned using Image Lab 5.0 (Bio-Rad, Hercules, CA, USA).
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