Similar to immunohistochemistry, after dewaxing, hydrating, repairing and incubation in 0.3 % Triton X-100 in PBS for 10 min, the donkey serum was incubated for 30 min, and the first anti-synaptopodin (1: 50, sc-515842, Santa), nephrin (1: 50, 66970-1, Proteintech), phosphorylated PI3K (p-PI3K, 1: 50, YP0765, immunoway) and phosphorylated Akt (p-Akt, 1: 50, YP0006, Immunoway) antibodies were incubated at 4 °C overnight. After being treated on a cover glass placed in 12-well plates, the MPC5 cells were fixed in 4 % paraformaldehyde for 10 min, and then incubated for 10 min with 0.3 % Triton X-100 at room temperature. MPC5 cells were incubated at 4 °C in the primary antibody after blocking with goat serum: synaptopodin (1: 50t, sc-515842Santa) and nephrin (1: 50, 66970-1-Ig, Proteintech). After PBS cleaning, the second antibody was incubated for 1 h at 37 °C with Alexa Fluor 488 or 594, followed by DAPI antifluorescence quenching tablets. Kidney sections and MPC cells were observed and photographed with a Leica laser confocal microscope.
Yp0006
Manufactured by Immunoway
Sourced in United States
YP0006 is a high-precision laboratory instrument designed for various analytical and research applications. It features advanced technology and robust construction to provide accurate and reliable results. The core function of YP0006 is to perform precise measurements and analyses, supporting a wide range of scientific investigations.
Automatically generated - may contain errors
Lab products found in correlation
Yt0177, by Immunoway (2 mentions)
Trizol, by Thermo Fisher Scientific (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Laser confocal microscope, by Leica (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Yt0470, by Immunoway (1 mentions)
S0002, by Affinity Biosciences (1 mentions)
Af7017, by Affinity Biosciences (1 mentions)
S0001, by Affinity Biosciences (1 mentions)
Af7003, by Affinity Biosciences (1 mentions)
Af5195, by Affinity Biosciences (1 mentions)
Af0199, by Affinity Biosciences (1 mentions)
Af7018, by Affinity Biosciences (1 mentions)
Af7022, by Affinity Biosciences (1 mentions)
Ripa buffer, by Beyotime (1 mentions)
Ab207442, by Abcam (1 mentions)
Ab133448, by Abcam (1 mentions)
Glucose detection kit, by Yuanye Bio-Technology (1 mentions)
Ab173305, by Abcam (1 mentions)
Cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions)
7 protocols using yp0006
1
Immunofluorescence Imaging of Podocyte Markers
2
Immunofluorescent analysis of liver tissue
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Paraffin-embedded and formalin-fixed liver tissue was sectioned at a thickness of 4 μM. The sections were deparaffinated in xylene-ethanol, washed 3 times in PBS for 5 min, antigen retrieval in sodium citrate buffer (0.01 mol/l, pH 6.8) for 20 min, washed 3 times in PBS for 5 min, placed in 0.3% H2O2 for 20 min, washed 3 times in PBS for 5 min, and incubated with 5% bull serum albumin in PBS/0.1% Triton X-100 for 60 min at 37°C. After draining the solution from the tissue section, the tissue was incubated with AKT (phospho Ser473) antibody (YP0006, Immunoway, USA) and Nrf2 antibody (AP52270, Abgent, USA), and then placed in a 4°C refrigerator overnight. After rinsing with PBS, the sections were incubated with FITC-conjugated secondary antibody for 120 min in 5% bull serum albumin. Nuclear staining was performed with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen), then mounted and photographed using a confocal laser scanning microscope (Axio Scope A1, Zeiss, Germany). The negative control experiments were also performed, in which the secondary antibodies or primary antibodies were omitted to exclude the potential non-specific binding to the tissue specimen.
3
Western Blot Analysis of Cellular Signaling
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Cells and aortas were lysed in RIPA buffer (Beyotime, Guangzhou, China), and 40 µg total protein was resolved in 12% or 10% SDS-PAGE gels and transferred onto polyvinylidene difluoride membrane, which was then blocked with 5% skimmed milk. Primary antibodies against iNOS (1:2,000, AF0199, Affinity, USA), COX-2 (1:2,000, AF7003, Affinity), CHOP (1:1,000, 2895, CST, USA), BCL-2 (1:1,000, YT0470, ImmunoWay, USA), BAX (1:1,000, YT0455, ImmunoWay), cleaved Caspase 3 (1:1,000, AF7022, Affinity), cleaved Caspase 8 (1:1,000, AF5267, Affinity), TIPE2 (1:1,000, DF3326, Affinity), TLR4 (1:2,000, AF7017, Affinity), TRIF (1:2,000, DF6289, Affinity), MyD88 (1:2,000, AF5195, Affinity), AKT (1:1000, YM3618, ImmunoWay), p-AKTSer473 (1:1000, YP0006, ImmunoWay), and β-actin (1:10,000, AF7018, Affinity) were used. HRP-conjugated corresponding secondary antibodies (1:10,000, S0001 and S0002, Affinity) were used, and antigen–antibody reactions were visualized using an enhanced chemiluminescence detection kit (E411-05, Vazyme, China). ImageJ 1.48 (NIH, Maryland, USA) was used to quantify the intensities of each band. β-actin served as an internal reference.
4
Investigating JQJTT's Effects on Diabetic Pathways
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JQJTT (190210) was provided by Longshunrong Pharmaceutical Factory of Tianjin Zhongxin Pharmaceutical Group Co., Ltd (Tianjin, China). PAL (CAS: 3486-67-7, purity >98%) was provided by Sichuan Vikqi Biotechnology Co., Ltd (Sichuan, China). FGF21 protein was obtained from Northeast Agricultural University. STZ and insulin were purchased from Sigma-Aldrich Co., Ltd (St. Louis, MO, United States). PD173074 was purchased from Selleck Chemicals (Houston, TX, United States). The glucose detection kit was obtained from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). Trizol and the cDNA reverse transcription kit were purchased from Thermo Fisher Scientific (Carlsbad, CA, United States). SYBR Green qPCR Mix was provided by Shandong Sikejie Biotechnology Co., Ltd (Shandong, China). Primary antibodies targeting phosphorylated (p)-FGFR1 (ab173305), FRS2 (ab137458), AMPK (ab207442), p-AMPK (ab133448), and β-actin (ab8227) were purchased from Abcam (Cambridge, United Kingdom); those targeting p-FRS2 (YP0805), p-AKT (YT0185), and AKT (YP0006) were obtained from ImmunoWay (California,United States); and that targeting FGFR1 (9740T) was purchased from CST (Boston, MA, United States). Secondary antibodies were obtained from Absin Biotechnology Co., Ltd (Shanghai, China).
5
Retinal Protein Expression Analysis
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Total protein was isolated from the retinal samples and run on 10% polyacrylamide gels following a standard protocol. Equal amounts of protein resolved by polyacrylamide gels were immunoblotted using antibodies against β-tubulin (1:10000, RM2003; Beijing Ray Antibody Biotech, Beijing, China), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:10,000, RM2002; Beijing Ray Antibody Biotech), caspase-3 (1:1000, 9662; Cell Signaling Technology, Danvers, MA, USA), Bax (1:1000, 2772; Cell Signaling Technology), B-cell lymphoma-2 (Bcl-2; (1:1000, ab182858; Abcam, Cambridge, UK), Akt (1:1000, YT0177; ImmunoWay, Plano, TX, USA), phosphorylated Akt (p-Akt; 1:1000, YP0006; ImmunoWay), glycogen synthase kinase-3 beta (GSK3β; 1:1000, 12456; Cell Signaling Technology), phosphorylated GSK3β (p-GSK3β; 1:1000, 9323; Cell Signaling Technology), and β-catenin (1:1000, ab32572; Abcam). They were then detected with a chemiluminescence kit. Gray value analysis was conducted using Image J software.
6
Western Blot Analysis of Protein Signaling
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Cells and tissues were lysed in RIPA buffer (Solarbio, Beijing, China), and proteins were separated by SDS-PAGE and transferred to polyvinyldi-fluoride membranes (Millipore, USA), and then the membranes were blocked with 5% blotting-grade milk for 1 h. the membranes were incubated overnight at 4°C with rabbit antibodies against CENPU (1:2,000, YN1585, Immunoway), rabbit antibodies against PI3K-p110α (1:1,000, YT3709, Immunoway), rabbit antibodies against AKT1 (1:1,000, YT0177, Immunoway), rabbit antibodies against pAKT Ser473 (1:1,000, YP0006, Immunoway), mouse antibodies against S6 (1:1,000, 2317S, Cell Signaling Technology), rabbit antibodies against pS6 Ser235/236 (1:1,000, YP0243, Immunoway), rabbit antibodies against NF-κB p65 (1:1,000, 8242S, Cell Signaling Technology), mouse antibodies against β-actin (1:1,000, 3700S, Cell Signaling Technology). The blots were then washed and incubated for 1 h at room temperature with peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG secondary antibody (Servicebio, GB23301 and GB23303, respectively, 1:3,000). The concentration of protein fragments was measured using Image-Pro Plus Ver. 7.0 software. URL: https://www.mediacy.com/imageproplus .
7
Protein Expression and Immunoblotting Analysis
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Proteins were lysed and extracted by RIPA buffer (89900, Thermo Scientific, United States). Subsequently, proteins were separated by SDS-PAGE and transferred to PVDF membranes. The primary antibodies used for immunoblotting were PI3K (1:4,000, YM3408, Immunoway, China), AKT (1:4,000, 4691, CST, United States), p-AKT (S473, 1:4,000, YP0006, Immunoway, China), P53 (1:4,000, Ab26, Abcam, China), P21 (1:2,000, ab109199, Abcam, China), P16INK4a (1:5,000, ab108349, Abcam, China), GAPDH (1:5,000, YM3029, Immunoway, China), and β-Actin (1:5,000, YM3028, Immunoway, China). The immunoreactive bands were visualized via electrochemiluminescence (ECL, DW101-01, TransGen Biotech, China) method and analyzed by ImageJ software (ver. 1.44, National Institutes of Health, United States).
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