Penile midshaft tissue was freshly harvested and divided into three parts transversally. One part was cryopreserved for protein extraction, one part was cryo-embedded for immunofluorescent (IHF) staining, and the final part was paraffin embedded for immunohistochemical (IHC) staining. For IHC and IHF, tissue sections used were 5 μm thick. Dilutions of primary antibodies (incubated overnight at 4°C) were as follows: alpha-smooth muscle actin (α-SMA; 1:3000; ab124964, Abcam, Cambridge, UK); neuronal-nitric oxide synthase (n-NOS; 1:500; ab95436, Abcam); neurofilament medium (NF; 1:500; ab7794, Abcam); and rat endothelial cell antigen-1 (Reca-1; 1:500; HIS52, Bio-Rad, Hercules, CA, USA). Appropriate species-directed secondary antibodies were applied to the sections for 90 min at room temperature; for IHF: α-SMA (1:500; A11012, Invitrogen, Carlsbad, CA, USA), n-NOS (1:500; A11001, Invitrogen); NF (1:500; A11012, Invitrogen), and Reca-1 (1:500; A11001, Invitrogen); and for IHC: α-SMA (ZB-2301, ZSGB-BIO, Beijing, China), nNOS (ZB-2305, ZSGB-BIO), NF (ZB-2301, ZSGB-BIO), and Reca-1 (ZB-2305, ZSGB-BIO). Semi-quantitative analysis was performed to evaluate the intensity of α-SMA, nNOS, NF, and Reca-1 staining using Image Pro Plus software (version 6.0, Media Cybernetics Corporation, Houston, TX, USA).
Zb 2301
Manufactured by ZSGB-BIO
Sourced in China, United States
The ZB-2301 is a benchtop centrifuge designed for general laboratory use. It features a digital display, a range of speed settings, and a compact footprint. The centrifuge is capable of handling various sample volumes and tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
Zb 2305, by ZSGB-BIO (49 mentions)
Ta 09, by ZSGB-BIO (14 mentions)
Pvdf membrane, by Merck Group (9 mentions)
Ipvh00010, by Merck Group (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Ripa lysis buffer, by Beyotime (6 mentions)
Ta 08, by ZSGB-BIO (5 mentions)
Bca protein assay kit, by Keygen Biotech (5 mentions)
Ripa buffer, by Beyotime (5 mentions)
Ab8226, by Abcam (5 mentions)
Polyvinylidene fluoride membrane, by Merck Group (4 mentions)
Ab133303, by Abcam (3 mentions)
Ap124p, by Merck Group (3 mentions)
Goat anti rabbit igg, by ZSGB-BIO (3 mentions)
D220014, by Sangon (3 mentions)
Ab0036, by Abways (3 mentions)
Image system, by Bio-Rad (3 mentions)
Chemiluminescence kit, by Merck Group (3 mentions)
Lysis buffer, by Keygen Biotech (3 mentions)
A11012, by Thermo Fisher Scientific (3 mentions)
134 protocols using zb 2301
1
Penile Tissue Histological Analysis
2
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (P0013B, Beyotime, Shanghai, China) with protease inhibitor (P0100, Solarbio life sciences, Beijing, China) was added to rat myocardial tissue or H9C2 cells to extract protein according to the manufacture’s protocol. The protein concentration was detected using a BCA Protein Assay Kit (P0012, Beyotime, Shanghai, China). Protein samples, which were mixed with loading buffer and heated at 100°C for 8 min, were separated by 12% SDS-PAGE gel electrophoresis and transferred to a polyvinylidene fluoride membrane (PVDF) pre-activated with methanol. The membrane was then blocked with 5% bovine serum albumin (BSA) for 1 h and immersed in the primary antibody solution overnight at 4°C. The primary antibodies were anti-NRF2, anti-HO-1 (E3F4S, CST, United States), anti-PKC (ab23511, Abcam, United Kingdom), anti-PKC (phosphor T497, ab59411, Abcam, United Kingdom), anti-α-tubulin (ab7291, Abcam, United Kingdom), and anti-H3 (17168-1-AP, Proteintech, Wuhan, China). The membranes were incubated with secondary antibodies (ZB-2301 or ZB-2305, ZSGB-BIO, Beijing, China) and the protein signals were detected using enhanced chemiluminescence (ECL) detection system. Images were analyzed using Image J software (National Institutes of Health, United States).
3
Immunohistochemical Analysis of AGE-RAGE Pathway
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We followed the methods of Wang et al. [21 (link)]. After blocking, the aorta sections were incubated overnight at 4°C with a rabbit anti-AGE antibody (1 : 200, ab23722, Abcam, Cambridge, UK), rabbit anti-RAGE antibody (1 : 200, ab3611, Abcam, Cambridge, UK), rabbit anti-Nox4 antibody (1 : 150, ab133303, Abcam, Cambridge, UK), mouse anti-3-nitrotyrosine antibody (1 : 200, ab61392, Abcam, Cambridge, UK), rabbit anti-NF-κB p65 antibody (1 : 200, D14E12, CST, Beverly, MA, USA), or mouse anti-Glo1 antibody (1 : 200, MA1-13029, Invitrogen, Waltham, MA, USA). The sections were washed and then incubated with a secondary antibody (1 : 200, peroxidase-conjugated anti-rabbit (ZB-2301) or anti-mouse (ZB-2305) antibody, ZSGB-BIO, Beijing, China) for 1 hour. Color was developed using DAB. Images were captured using an Olympus DP71 microscope. The mean IOD of staining (IOD/area) from 5 random fields on one section was assessed using Image-Pro Plus 6.0 software.
4
Protein Expression Analysis Protocol
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The total protein extraction kit was purchased from KeyGENBioTECH (Jiangsu, China) and the protein quantification kit by BCA method was purchased from LEAGENE (Beijing, China). The target proteins of different molecular weights were separated by 10% polyacrylamide gel electrophoresis and transferred to the solid phase carrier PVDF membrane. Incubated primary antibody ADORA2B (1:1000, ER1903-44, HUABIO, China) ,β-catenin (1:1000, ab32572, Abcam, USA) ,N-cadherin (1:2000, ab18203, Abcam, USA) ,vimentin (1:2000, 10366-1-AP, Proteintech, China), and E-cadherin (1:2000, ab40772, Abcam, USA) overnight at 4°C and incubated horseradish peroxidase labeled rabbit secondary antibody (1:5000, Zb-2301, ZSGB-BIO, China) for 1h. After 3 times of TBST washing, the protein bands were displayed by ECL luminescence solution and autoradiography. Finally, the gray values were analyzed by ImageJ software.
5
Quantifying Endoplasmic Reticulum Stress Response
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The aorta tissue or RAW264.7 macrophages were lysed for 30 min at 4°C in lysis buffer. Total protein concentrations were determined using bicinchoninic acid reagent. Total protein (50–100 μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes. After blocking, the membranes were incubated with anti-IRE1α (ab37073; 1:800; Abcam), anti-glucose-regulated protein 78 (GRP78; 11587-1-AP; 1:500; ProteinTech, Wuhan, China), anti-p-IRE1α (Ser724; ab48187; 1:800; Abcam), IκB kinase α (IKKα; 2682S; Cell Signaling Technology, Danvers, MA, USA), IκB kinase β (IKKβ; 2684S; Cell Signaling Technology), anti-p-IκB kinase α/β (p-IKK; Ser176/180; 2697T; 1:800; Cell Signaling Technology), IκB (YM3718; 1:500; ImmunoWay, Newark, DE, USA), anti-NF-κB p65 (YT5339; 1:500; ImmunoWay), anti-β-actin (6008-1-Ig; 1:750; ProteinTech) and anti-LaminB (sc-6216; 1:500; ZSGB-Bio) primary antibodies overnight at 4°C. Horseradish peroxidase-conjugated secondary antibodies (ZB-2301; ZSGB-Bio) were applied the following day for 2 h at 37°C. The bands were visualized using chemiluminescence (Beyotime, Shanghai, China). Band density was analyzed using fusion software (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and normalized to β-actin or Lamin B density.
6
Western Blot Protein Analysis Protocol
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Western blot analysis was done according to the method described by Yin et al. [18 (link)]. Protein extracts were prepared in RIPA Lysis Buffer (Beyotime Biotechnology Inc., Shanghai, China). Approximately 30 μg proteins were subjected to SDS-PAGE electrophoresis. The duration of the electrophoresis was 30 V for 30 min, 80 V for 30 min, and 120 V for 30 min. Then, the proteins were transferred onto a PVDF membrane (Millipore, MA, USA) and blocked with 5% non-fat milk in Tris-Tween buffered saline for 1.5 h. Anti-Metallothionein rabbit polyclonal to antibody (ab233289, 1:5000, Abcam) and anti-beta actin mouse monoclonal antibody (60008-1, 1:5000, Proteintech) were used. The antibodies were incubated for 12 h at 4 °C. After incubation with the HRP-conjugated secondary antibodies (ZB-2301, 1:5000, ZSGB), the bands were visualized using the Alpha Imager 2200 software (Alpha Innotech Corporation, San Leandro, CA, USA).
7
DNA Methylation Detection Assay
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DNA samples were denatured at 95 °C for 10 min, snap-cooled on ice and neutralized with 10% volume of 6.6 M ammonium acetate. Samples (2 μL) were spotted on the membrane (Amersham Hybond-N+, GE), air-dried and then UV-cross-linked (2× auto-crosslink, 1800 UV Stratalinker, STRATAGENE). Membranes were blocked in blocking buffer (5% milk, PBS-T) for 2 h at room temperature, and incubated with N6-mA antibodies (202–003, Synaptic Systems, 1:1000) overnight at 4 °C. After 5-time washes, membranes were incubated with HRP-linked secondary anti-rabbit IgG antibody (ZB-2301, ZSGB-BIO, 1:5000) for 1 h at room temperature. Signals were detected with Immun-StarTM WesternCTM Chemiluminescence (Bio-Rad).
8
Immunohistochemical Analysis of GPX2 and ABCB6 Expression
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Formalin-fixed, paraffin-embedded tissues were cut to generate consecutive 4 μm sections, which were then subjected to IHC analysis. The sections were incubated with antibodies against GPX2 (catalogue no. ab140130, Abcam; 1 : 200 dilution) and ATP binding cassette subfamily B member 6 (ABCB6) (b224575, Abcam; 1 : 200 dilution) at 4°C overnight. After washing in PBS, sections were further incubated with a HRP-conjugated secondary antibody (catalogue no. ZB-2301, Zsbio; 1 : 500 dilution) for 30 min at 37°C. Then, the tumor tissues were incubated with a substrate-chromogen (DAB) solution for 10 min. Finally, automated hematoxylin was used to counterstain the slides for 5 min. The immunostaining was microscopically evaluated by two independent pathologists. A semiquantitative scoring system was used that is based on the staining intensity and the proportion of positive cells [13 (link)].
9
Western Blotting Protocol for Protein Analysis
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Western blotting was performed as previously reported [25 (link)]. Equal amounts of protein (up to 30 μg) from the treated mice were separated by 10% SDS–PAGE and transferred to PVDF membranes. Transferred blots were blocked with nonfat milk for 2 hours and then incubated overnight at 4 °C with primary antibody (1:1000). Blots were subsequently washed and incubated with goat anti-rabbit (ZB-2301, Zsgb-Bio, 1:5000) and goat anti-mouse (ZB-2305, Zsgb-Bio, 1:5000) secondary antibodies for 2 hours. The antibodies were listed in Table S1 . Protein bands were detected with ECL reagent (WBKLS0500, Merck Millipore). Chemiluminescent signals were detected and analyzed using a Tanon-5500 chemiluminescent imaging system (Tanon Science and Technology Co., Ltd., Shanghai, P R China). The intensity of the bands was analyzed using ImageJ 1.49 software (National Institutes of Health, Bethesda, MD, USA). Full-length blots/gels are presented in Supplementary Fig. S7 .
10
QPRT knockdown protein expression
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The QPRT knockdown (QPRT siRNA), negative control (QPRT siRNA NC), and wildtype (control) HEK293T cells were seeded in 6-well plates. Cells were lysed in lysis buffer for 30 min on ice, and then insoluble materials were removed after 15 min centrifugation at 10,000 rpm/min. Protein concentrations were determined by BCA assay (Pierce, Rockford, United States). Lysate supernatant was separated using 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (IPVH00010, Millipore). The membrane was incubated with Rabbit monoclonal antibody against QPRT (ab171944; Abcam) or Mouse Anti-GAPDH antibody (1/2000; TA-08; ZSGB-BIO) followed by incubation with peroxidase-conjugated goat anti-rabbit IgG (H + L) (1/2000; ZB-2301; ZSGB-BIO) and peroxidase-conjugated goat anti-mouse IgG (H + L) (1/2000; ZB-2305; ZSGB-BIO) secondary antibody, respectively. Western blots were developed using ECL solution. Grayscale results were analyzed by “Quantity one” software.
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