Cell lines stable expressing vector or pcDNA3.1-MT1M were washed with ice-cold PBS for 3 times and treated with lysis buffer. Protein sample (40 μg) was separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and immune-stained as the manufactory instruction. Bands were detected through ECL detection system. The primary antibodies were used as follows: anti-MT1M antibody (#ab158927; Abcam, USA), PCNA（#ab92592; Abcam), E-cadherin (#ab231303; Abcam), N-cadherin (#ab76011; Abcam), Vimentin (#ab8069; Abcam), SOD1 (#ab51254; Abcam), SOD2 (#13194; Cell Signaling Technology, USA), SOD3 (sc-271170; Santa Cruz Biotechnology, USA), Erk (#4372; Cell Signaling Technology), p-Erk (sc-7383; Santa Cruz Biotechnology), Akt (#4685; Cell Signaling Technology), Nrf2 (sc-365949; Santa Cruz Biotechnology), GPx2 (ab137431; Abcam), p-Akt (sc-4060; Santa Cruz Biotechnology), rabbit polyclonal anti-PI3 kinase p85 alpha (phosphor-Y607, #ab182651; Abcam), cleaved caspase-3 (#9661; Cell Signaling Technology), bcl-2 (#ab32124; Abcam)，BAX (#9942; Cell Signaling Technology), and GAPDH (bsm-51010M; BIOSS, China) as a control. The dilution of primary and secondary antibodies was used according to the instructions. The signals were detected with an enhanced chemiluminescence kit (Amersham Pharmacia Biotech, USA) and the band intensities were quantified by Image-Pro Plus 6.0 (Bio-Rad, USA).
Image pro plus 6
Manufactured by Bio-Rad
Sourced in United States
Image-Pro Plus 6.0 is a software application designed for image analysis and processing. It provides tools for capturing, enhancing, measuring, and analyzing digital images from a variety of sources, including microscopes and other imaging devices.
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Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Chemidoc imaging system, by Bio-Rad (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Enhanced chemiluminescence kit, by Cytiva (1 mentions)
Vimentin, by Abcam (1 mentions)
Bcl 2, by Abcam (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Gapdh, by Bioss Antibodies (1 mentions)
E cadherin, by Abcam (1 mentions)
N cadherin, by Abcam (1 mentions)
P erk, by Santa Cruz Biotechnology (1 mentions)
Hrp conjugated goat anti rabbit igg h l secondary antibody, by Thermo Fisher Scientific (1 mentions)
Rabbit anti β actin, by Cell Signaling Technology (1 mentions)
Rabbit anti inducible nitric oxide synthase, by Cell Signaling Technology (1 mentions)
Tcs sp2, by Leica (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Rankl, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
Phospho stat3 tyr705, by Cell Signaling Technology (1 mentions)
β actin, by Huabio (1 mentions)
11 protocols using image pro plus 6
1
Western Blot Analysis of MT1M and EMT Markers
2
Quantifying Hippocampal Inflammation in AD/TBI Mice
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The hippocampus was dissected out from APP/PS1 and APP/PS1 TBI mice. Western blotting was performed as described previously (30 (link)). Target protein expression was measured using the following primary antibodies: rabbit anti-arginase-1 (Arg1) (catalog number: #93668; Cell Signaling Technology), rabbit anti-inducible nitric oxide synthase (iNOS) (catalog number: #13120; Cell Signaling Technology), and rabbit anti-β-actin (catalog number: #4790; Cell Signaling Technology) (IgGs; 1:2000). β-Actin was utilized as an internal reference. The protein blots were visualized using an HRP-conjugated goat anti-rabbit IgG (H + L) secondary antibody (1:5000, Invitrogen; Thermo Fisher Scientific) and a Chemi-Doc™ Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and analyzed using Image-Pro Plus 6.0.
3
Quantifying Myocardial Collagen Fibrosis
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The quantity of myocardial collagen fibrosis on sections stained with Sirius Red was measured by Image Pro Plus 6.0 (GelDoc2000, Bio-Rad). In each section, digital photographs were taken at five randomly selected fields. The myocardial fibrosis area percentage was calculated by the ratio of the stained area to the total area. The calculation of the collagen volume fraction is as follows: collagen volume fraction (CVF, %) = (total area of collagen/total area of image) × 100%.
4
Allergic Response Evaluation Protocol
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OVA (Sigma A8040, USA) was used as antigen, and aluminum hydroxide as adjuvant (lot No. 201110328, Chengdu Kelong Chemical Factory). The No. 9 needle (YZB/Su0313-2007, Yangzhou Co. Ltd., China), and the No. 000 catgut (YY1116-2002, Shanghai Co. Ltd., China) are disposable applications. sIgE, IL-4, and IFN-γ enzyme-linked immunosorbent assay (ELISA) kits were purchased from Abcam (Yonghui Bio Co. Ltd., Beijing, China). The SP and TLR2 polyclonal antibody (rabbit anti-mouse) and TLR4 monoclonal antibody (mouse anti-rat) were from Abcam (Trading Co. Ltd., Shang Hai, China). Sodium citrate buffer (0.01 M, pH 6.0) was prepared for dilution. Microscopic image acquisition and analysis system (MikeAudiBA200 Digital and Image-Pro Plus 6.0, USA) and ChemiDoc XRS gel imaging apparatus (Bio-Rad, USA) were purchased from the respective manufacturers.
5
Immunofluorescence Staining of Mouse Brain Tissue
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Mice were humanely killed 3 weeks after injury, and their brain samples were prepared as previously described [11 (link)]. Briefly, mice were perfused with 0.9% saline and 4% paraformaldehyde successively through the left ventricle after being deeply anesthetized with 5.0% isoflurane. Then, the brains were removed, fixed, dehydrated in methanol, and embedded in paraffin and 20 μm slices cut using a freezing microtome. The immunofluorescence staining protocol has been previously described in detail [11 (link)]. In brief, the brain slices were fixed in methanol for 10 min at -20°C, and then, 10% BSA was used for blocking at room temperature for 1 h. Finally, the brain slices were incubated with the primary antibodies Bcl-2 and Bax (1 : 200), GAP-43 (1 : 200), and SYN (1 : 150) at 4°C overnight. Then, brain slices were incubated with secondary antibodies (anti-mouse IgG, 1 : 3,000) at room temperature after being thoroughly washed with PBS. All sections were viewed with a confocal microscope (Leica TCS SP2, Germany) using the same parameters. Four visual fields (400x) were randomly selected for each brain slice and digital images captured. All images were analyzed using Image-Pro Plus 6.0 (Bio-Rad, CA, USA).
6
Western Blot Analysis of Protein Expression
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At the end of the iPTH treatment, the total protein was extracted using the Total Protein Extraction Kit (Signalway Antibody, USA), the Nuclear protein was extracted using a Nucleoprotein Extraction Kit (Sangon Biotech, China). Western blotting was conducted as previously described.62 (link) Briefly, the protein concentration was determined using a BCA Protein Assay Kit (Beyotime, China). After denaturation, the samples were loaded onto SDS-polyacrylamide gel electrophoresis gels for electrophoresis and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% skim milk or 5% BSA and incubated with primary antibodies against β-actin (Huabio, China), STAT3 (Huabio, China), phospho-STAT3 (Tyr705) (Cell Signaling Technology, USA), RUNX2 (Huabio, China), OSX (Abcam, UK), ALP (Huabio, China), OPN (Huabio, China), β-catenin (Huabio, China), RANKL (Abcam, UK) and OPG (Huabio, China) at 4 °C overnight, and rabbit and mouse secondary antibodies (Signalway Antibody, USA). The bands were visualized with a ChemiDoc Touch Imaging System (Bio-Rad, USA) and quantified with Image-Pro Plus 6.0.
7
Western Blot Analysis of Liver Proteins
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Liver tissues were lysed in sample buffer containing 62 mM Tris-HCl, pH 6.8, 0.1% SDS, 0.1 mM sodium orthovanadate, and 50 mM sodium fluoride. The protein content was determined by the BCA protein assay. Equal amounts of proteins were loaded and separated by SDS-PAGE. After electrophoresis, the proteins were transferred on membranes, after being blocked with 3% nonfat dry milk the membrane with target proteins was incubated with an antibody against IRS-2, SREBP-1c, ACC, FAS, or GAPDH overnight at 4°C. The blots were incubated with a respective HRP-conjugated second antibody, and then immunoreactive bands were revealed using an enhanced chemiluminescence system (Applygen Technologies Inc., Beijing, China). The protein signal was quantified by scanning densitometry in the X-film by Image-Pro Plus 6.0 software (Bio-Rad, Hercules, California, USA) [24 (link)].
8
Western Blot Analysis of Testicular Proteins
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The testicular tissues were extracted and transferred into a centrifuge tube containing RIPA lysis buffer supplemented with phenylmethanesulfonyl fluoride (PMSF). Subsequently, the homogenate was subjected to centrifugation at 12,000× g for 5 min at 4 °C. Following the collection of the Western blot supernatant, a BCA protein assay kit was utilized to ascertain the total protein concentrations. Each sample, containing 20 mg of lysate, was loaded onto a 12% SDS-PAGE gel for electrophoresis and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). For Western blotting, proteins were first separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto PVDF membranes (Millipore, Bedford, MA, USA). After blocking with a 5% nonfat milk–TBST buffer for 1 h at 4 °C, the membranes were incubated overnight at 4 °C with primary antibodies, including GAPDH, caspase-3, cleaved-caspase-3, Bax, Bcl-2, STAT3, p-STAT3, JAK2, and p-JAK2. Following incubation, the membranes were washed three times with TBST at room temperature and then incubated with the corresponding secondary antibody for 1 h. Finally, protein bands were visualized using an ECL reagent and quantitatively analyzed with Image-Pro Plus 6.0 software (BioRad, Hercules, CA, USA).
9
Testicular Protein Expression Analysis
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The testicular tissues were removed and placed into a centrifuge tube containing RIPA lysis buffer and phenylmethanesulfonyl fluoride (PMSF), and the homogenate was centrifuged at 12,000 rpm for 5 min at 4 °C. After collecting the supernatants for western blots, a BCA protein assay kit was used to determine the total protein concentrations. Sample proteins were separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Millipore, Bedford, MA, USA). After blocking with a 5% nonfat milk–TBST buffer for 1 h at 4 °C, the membranes were incubated with the following primary antibodies overnight at 4 °C: Bax, Bcl-2, caspsae-3, STAT3, p-STAT3, JAK2, and p-JAK2. After incubation, the membranes were washed three times with TBST at room temperature and incubated with the appropriate secondary antibody for 1 h. Finally, the protein bands were visualized using ECL reagent, and were quantitatively analyzed using Image-Pro Plus 6.0 software (BioRad, Hercules, CA, USA).
10
Adipose Tissue Protein Extraction and Western Blotting
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Details of the western blotting procedure were reported previously (Batistel et al., 2017; (link)Liang et al., 2021) (link). Briefly, approximately 100 of mg adipose tissue was homogenized in 1 mL of RIPA Lysis and Extraction Buffer (catalog no. 89900, Thermo Fisher) containing Halt protease and phosphatase inhibitor cocktail (100×, catalog no. 78442; Thermo Fisher Scientific) for 60 s and then placed on ice for 20 min. The lysate was centrifuged at 14,000 × g for 15 min at 4°C. Protein concentration of the supernatant was determined using a Pierce Bicinchoninic Acid Protein Assay Kit (catalog no. 23227, Thermo Fisher Scientific). Protein samples were denatured by heating at 95°C for 5 min before loading 20 μL of protein into each lane of 4% to 20% Mini-Protean TGX precast gels (catalog no. 4561093, Bio-Rad) and transferred onto a polyvinylidene difluoride membrane (catalog no. 1620261, Bio-Rad) in a The enhanced chemiluminescence signals were acquired using an imaging system (ChemiDoc MP, Bio-Rad), and the intensities of the bands were analyzed using Image-Pro Plus 6.0 software (Bio-Rad). All information for primary antibodies is listed in Supplemental Table S3.
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