For polychromatic immunofluorescence, the Opal Multiplex IHC Assay Kit (PerkinElmer, USA) was employed on formalin-fixed paraffin-embedded FIA sections following the manufacturer's instructions. Fluorescent images were acquired using a Zeiss Axio Scope A1 microscope. Primary antibodies included: anti-MMP-9 (1:20, BM4089, Boster), anti-α-SMA (1:20, BM3902, Boster), anti-ICAM1(1:50, A00171, Boster), anti-p-JAK2 (1:500, bs-3206R, Bioss), anti-p-Src (1:50, bs-1730R, Bioss), anti-p-PLCγ (1:100, #MAB74542, R&D systems), anti-p-Erk1/2 (1:100, bs-3016R, Bioss), anti-p-STAT1 (1:50, #7649, CST), anti-p-STAT3 (1:20, BM4835, Boster). Rabbit IgG (#3900, CST) or mouse IgG1 (#5415, CST) with the same concentration as primary antibodies were utilized as isotype controls to validate antibody specificity. Meanwhile, secondary antibody only controls were used to distinguish genuine target staining from background.
Bs 3016r
Manufactured by Bioss Antibodies
Sourced in China
The Bs-3016R is a laboratory equipment product manufactured by Bioss Antibodies. It is a device used for laboratory analysis and research purposes. The core function of the Bs-3016R is to facilitate precise measurements and data collection during scientific experiments and investigations.
Automatically generated - may contain errors
Lab products found in correlation
Bs 0439r, by Bioss Antibodies (2 mentions)
Bs 5477r, by Bioss Antibodies (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Opal multiplex ihc assay kit, by PerkinElmer (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
Bm4089, by Boster Bio (1 mentions)
Bm3902, by Boster Bio (1 mentions)
Blotting system, by Bio-Rad (1 mentions)
Ecl plus western blotting detection system, by Cytiva (1 mentions)
M pertm mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Yt0099, by Immunoway (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Bs 0636r, by Bioss Antibodies (1 mentions)
Bs 17591r, by Bioss Antibodies (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Enhanced chemiluminescence substrate, by Merck Group (1 mentions)
Proteinase inhibitor, by Merck Group (1 mentions)
Bs 0061r, by Bioss Antibodies (1 mentions)
Bs 0587r, by Bioss Antibodies (1 mentions)
8 protocols using bs 3016r
1
Multiplex Immunofluorescence Analysis
2
Protein Extraction and Western Blot Analysis
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For protein extraction, heterophils were incubated with 40 μM FB1 in white, clear-bottomed 6-well plates for 1.5 h at 37˚C with 5% CO2. Then cells were lysed by M-PERTM Mammalian Protein Extraction Reagent (Thermo Scientific, Rockford, IL) for 7 min at 37˚C. The lysates (n = 3) were collected into clean 1.5 mL centrifuge tubes and centrifuged at 12,000 rpm for 10 min at 4˚C, and protein concentrations were measured with Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL).
Each sample with protein (40 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and moved onto polyvinylidene difluoride membrane for 45 min at 85V in blotting system (Bio-Rad, Singapore). Membranes subsequently were blocked with 5% Blotting Grade (dissolved in TBST) and incubated with polyclonal antibody to β-actin (YT0099, Immunoway Biotechnology Company, Plano, TX), p38 (8690S, Cell Signaling Technology, Danvers, MA), p-p38 (4511S, Cell Signaling Technology, Danvers, MA), ERK (BM4326, BOSTER, China), or p-ERK (bs-3016R, BIOSS, China) at 4˚C overnight. Next, the membranes were incubated with the corresponding HRP-linked secondary antibodies. Finally, the blots were detected by the ECL Plus Western Blotting Detection System (Amersham Life Science, UK).
Each sample with protein (40 μg) were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and moved onto polyvinylidene difluoride membrane for 45 min at 85V in blotting system (Bio-Rad, Singapore). Membranes subsequently were blocked with 5% Blotting Grade (dissolved in TBST) and incubated with polyclonal antibody to β-actin (YT0099, Immunoway Biotechnology Company, Plano, TX), p38 (8690S, Cell Signaling Technology, Danvers, MA), p-p38 (4511S, Cell Signaling Technology, Danvers, MA), ERK (BM4326, BOSTER, China), or p-ERK (bs-3016R, BIOSS, China) at 4˚C overnight. Next, the membranes were incubated with the corresponding HRP-linked secondary antibodies. Finally, the blots were detected by the ECL Plus Western Blotting Detection System (Amersham Life Science, UK).
3
Protein Expression Analysis in Lung Tissue
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Lung tissues were homogenized in RIPA lysis buffer (Beyotime, China) with protease inhibitors, followed by centrifugation at 12,000 rpm at 4 °C, and the supernatants were collected. The proteins were separated in 8–10% sodium dodecyl sulfate-poly-acrylamide gels and transferred to polyvinylidene fluoride membranes. The membranes were cut prior to hybridization with antibodies. Next, the membranes were incubated with primary antibodies against ACE (1:1,000; #bs-0439R, Bioss, Beijing, China), ACE2 (1:1,000, #21,115–1-Ig, Proteintech, China), ATIR (1:1,000; #bs-0630R, Bioss, Beijing, China), p38 (1:500 #OM125772 OmnimAbs China), p-p38 (#bs-0636R 1:1,000 Bioss), JNK (#OM260186, 1:500, OmnimAbs), p-JNK (#bs-17591R, 1:1,000, Bioss), ERK1/2 (#OM125780, 1:500, OmnimAbs, China), p-ERK1/2 (#bs-3016R, 1:1,000, Bioss), NF-κB p65 (#66,535–1-lg, 1:1,000, PTG), and p-NF-κB p65 (#bs-3485R, 1:1,000, Bioss) at 4 °C overnight. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000, #bs0295M, Bioss, Beijing, China) for 1 h at room temperature. Finally, the proteins were visualized using ECL reagents (Millipore, USA) and analyzed using ImageJ.
4
Protein Expression Analysis of Renin-Angiotensin System
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Lung and heart tissues were lysed with a buffer containing RIPA (Beyotime, China), 10 % phosphatase inhibitor (Roche Applied Science, Germany) and 1 % proteinase inhibitor (Sigma, USA). Bicinchoninic acid assay was used to measure the protein concentration. Equal amounts of the samples were loaded on 10 % SDS–PAGE gels. Then, protein was transferred to a PVDF membrane (Millipore Corporation, USA) and incubated for 1 h at room temperature in blocking solution (5 % non-fat milk). The membrane were incubated overnight at 4 °C in blocking solution containing primary antibodies. Then, it was washed and incubated with horseradish peroxidase conjugated secondary antibody (Cell Signaling Technology, USA) for 1 h at room temperature. After a second wash, the membrane was developed using enhanced chemiluminescence substrate (Millipore Corporation, USA). The band intensities were analyzed using Image J software (National Institutes of Health, Bethesda, USA). Primary antibodies against renin, Ang II, ACE, mineralocorticoid receptor (MR), BNP, phospho-ERK1/2 (Thr202 + Tyr204) and β-actin were purchased from BIOSS Biotechnology (bs-6184R, bs-0587R, bs-0439R, bs-1850R, bs-7132R, bs-3016R, bs-0061R). Primary antibodies against Ang II type 2 receptor (AT2 receptor) and ANP were obtained from Santa Cruz Biotechnology (sc-9040, sc-18811).
5
Liver Protein Quantification and Western Blot Analysis
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The protein was extracted from liver tissues, and protein concentration was determined using the bicinchoninic acid assay. Briefly, 20 μg protein in each group was processed in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis. After transferring onto polyvinylidene fluoride (PVDF) membrane, non-specific staining was blocked by 5% skim milk. The membrane was incubated with the following antibodies overnight at 4°C: rabbit anti-PBEF (1:2000, 11776-1-AP, Proteintech); rabbit anti-ERK1/2 (1:1000, 16443-1-AP, Proteintech); rabbit anti-p-ERK1/2 (1:500, bs-3016R, Bioss); rabbit anti-p38MAPK (1:1000, bs-0637R, Bioss); rabbit anti-p-p38MAPK (1:1000, bs-5477R, Bioss); rabbit anti-AKT (1:500, ab8805, Abcam); rabbit anti-p-AKT (1:1000, bs-2720R, Bioss); rabbit anti-AQP1 (1:1000, bs-1506R, Bioss); rabbit anti-AQP5 (1:1000, AF5169, Affinity); rabbit anti-ENaC (1:1000, bs-2957R, Bioss); rabbit anti-surfactant protein D (SP) (1:1000, ab220422, Abcam); and mouse monoclonal anti-GAPDH (1:2000, TA-08, ZSBio).
6
Western Blot Analysis of Cellular Proteins
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Total protein was extracted from cells using RIPA buffer (Beyotime), with an equal amount loaded onto 12% SDS‐PAGE gel. The following primary antibodies were used for Western blot analysis: Suv39h1 (48 kD, 1:800; Abcam, ab12405), proliferating cell nuclear antigen (PCNA, 29 kD, 1:300; Bioss, bs‐0754R), complement C3 (180 kD, 1:200; Santa Cruz, sc20137, CA), phosphor‐p38 (Tyr323) (41 kD, 1:300; Bioss, bs‐5477R), phosphor‐ERK1/2 (Thr202 + Tyr204) (41 kD, 1:300; Bioss; bs‐3016R), p38 (41 kD, 1:300; Bioss, bs‐0637R), ERK1/2 (42/44 kD, 1:300; Bioss, bs‐2637R), histone H3K9 trimethylation (H3K9me3, 15.4 kD, 1:800; Abcam, ab8898) and GAPDH (37 kD, 1:1000; Cell Signaling, #5174). Following the incubation with primary antibodies, HRP‐conjugated secondary antibodies (1:1500‐1:2000; Boster) were used. The primary antibody was diluted with Tris‐buffered saline Tween‐20 (TBST) containing 5% skimmed milk powder.
7
Evaluating Anti-Inflammatory Agents
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Rabbit anti-ERK1 + ERK2 antibody (bs-0022R), rabbit anti-phospho-ERK1/2 antibody (bs-3016R), rabbit anti-p38 MAPK antibody (bs-0637R), rabbit anti-Phospho-p38 MAPK (Thr180 + Tyr182) antibody (bs-0636R), rabbit anti-JNK1 + JNK2 + JNK3 (bs-2592R) and rabbit anti-phospho-JNK1 + JNK2 + JNK3 (T183 + T183 + T221) (bs-4163R) antibody were obtained from Beijing Biosynthesis Biotechnology Co., Ltd. Compound dexamethasone acetate cream was obtained from Guangzhou Baiyunshan Pharmaceutical Group Co., Ltd. Curcumin, sesamin, berberine, coptisine hydrochloride, phellodendrine, and ferulic acid standards with a purity greater than 98% were obtained from the China National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China. Assay kits for IgE, IL-4, tumor necrosis factor (TNF)-α, IL-6, IL-2, and interferon (INF)-γ were purchased from RD, USA.
8
Immunohistochemistry and Western Blot Analysis of ERK1/2 and pERK1/2
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The primary antibodies were utilized in this study, including the rabbit polyclonal anti-ERK1/2 (BS-2637R, Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), rabbit polyclonal anti-pERK1/2 (BS-3016R, Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), and β-actin (BS-0061R, Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China). The dilution of ERK1/2 and pERK1/2 antibodies for immunohistochemistry and Western blot observations were respectively 1:200 and 1:750, and the dilution of β-actin antibodies for Western blot observations was 1:1000.
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