The protein expression of SD rat BMSCs was detected by Western Blotting. After 24 h, the medium was replaced with normal osteogenic induction medium and osteogenic induction medium containing 10% of different scaffold extracts. After culturing for 14 days, cell precipitation was collected; 100 µl of RIPAcell lysate (Beyotime, China) containing 1 mM PMSF, (Beyotime, China) was added to each 6-well plate, lysed, centrifuged, and collected the supernatant. Total cellular protein was quantified using a BCA protein assay kit. Then the samples were subjected to SDS electrophoresis and transferred to a polyvinylidene fluoride membrane. The membranes were first blocked with 5% skim milk for 1 h, and then incubated at 4 °C overnight with primary antibodies as follows: RUNX2 (AF5186, Affinity, 1:500), OCN (DF12303, Affinity, 1:500), ALP (DF6225, Affinity, 1:500), and Collagen I (AF7001, Affinity, 1:500). After being washed with TBST three times, the membranes were incubated with HRP-conjugated secondary antibodies (1:10,000). The antigen–antibody complex was visualized. Signal intensities were quantified using ImageJ software.
Af7001
Manufactured by Affinity Biosciences
Sourced in United States, China, United Kingdom
The AF7001 is a laboratory centrifuge designed for general-purpose applications. It features a compact design and a maximum rotational speed of 6,000 RPM. The centrifuge can accommodate a variety of rotor types and sample volumes, making it a versatile instrument for a range of laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Af0136, by Affinity Biosciences (6 mentions)
Af1032, by Affinity Biosciences (5 mentions)
Ripa buffer, by Beyotime (5 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (3 mentions)
Af5186, by Affinity Biosciences (2 mentions)
Df6225, by Affinity Biosciences (2 mentions)
Df12303, by Affinity Biosciences (2 mentions)
Af5103, by Affinity Biosciences (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Ab32575, by Abcam (2 mentions)
Af7021, by Affinity Biosciences (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Ripa cell lysate, by Beyotime (1 mentions)
Ab124964, by Abcam (1 mentions)
Enhanced chemiluminescence plus kit, by Cytiva (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Bsm 52214r, by Bioss Antibodies (1 mentions)
Gb12002, by Wuhan Servicebio Technology (1 mentions)
Ab76026, by Abcam (1 mentions)
Ab28947, by Abcam (1 mentions)
35 protocols using af7001
1
Evaluating Osteogenic Potential of Rat BMSCs
2
Immunohistochemical Analysis of Collagen I
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Tissue blocks smaller than 0.5 cm × 0.5 cm × 0.1 cm were taken for standby, then washed five times with phosphate-buffered saline (PBS). IHC was performed according to standard protocols. Briefly, after fixation, embedding, slicing, and dewaxing, antihelion was repaired with 0.01 M sodium citrate solution for 20 min and the endogenous peroxidase activity was quenched with 3% H2O2 for 10 min. The tissues were then left in 1% serum in PBS at 37 °C for 20 min to eliminate nonspecific binding. Following the blocking, the sections were rinsed and incubated overnight at 4 °C with collagen I antibody (AF7001, Affinity Biosciences, Beijing, China). Thereafter, biotinylated rabbit secondary antibody (SP-9001, ZSJQ-BIO, Guangdong, China) was added to the slices and left for 20 min at 37 °C. Horseradish enzyme, labeled streptomyces ovalbumin working solution (SA/HRP), was added to the slices at left at 37 °C for 20 min. The sections were developed by conventional dehydration, tissue clearing, diaminobenzidine (DAB) staining, and neutral resin sealing. Image-Pro Plus 6.0 software was used for assessing results and expressed as a percentage of the total analyzed areas.
3
Kidney Tissue Protein Expression Analysis
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We carried out western blot analyses to detect target protein levels in kidney tissues and cells. The primary antibodies included polyclonal antibodies against phospho-YAP (Ser127) (p-YAP, 1:1000, bsm-52214R, Bioss, China), YAP (1:1000, #14074, Cell Signaling Technology, United States), α-SMA (1:10000, ab124964, Abcam, United States), collagen Ⅰ (1:750, AF7001, Affinity, United States), and GAPDH (1:1000, GB12002, Servicebio Technology, China). The primary proteins were detected by using horseradish peroxidase-conjugated secondary antibodies (Abcam, United States), and the immune complexes were finally developed by using the enhanced chemiluminescence Plus kit (Amersham, Freiburg, Germany).
4
Immunofluorescence Staining of E-Cadherin, α-SMA, and Collagen I
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For immunofluorescence staining, the sections were blocked with goat serum and incubated with E-Cadherin (1: 200) and ɑ-SMA (1: 100) antibodies and collagen I (AF7001, Affinity Biosciences, Cincinnati, OH, USA, 1: 200) at 4°C overnight. Then the specimens were incubated with DAPI and secondary antibodies. Finally, the reaction was visualized using a fluorescence microscope.
5
Tissue Analysis Protocol for Histochemistry and IHC
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Tissue specimens were fixed in 10% formalin and embedded in paraffin. Tissue samples were cut at 5 µm thicknesses, and then sections were deparaffinized and rehydrated, followed by hematoxylin and eosin (H&E) and Masson's trichrome staining. Stained sections were imaged under a microscope. Immunohistochemical staining was performed according to the standard protocols. Sections were incubated with the following antibodies: p‐Nrf2 (Ab76026, Abcam), HO‐1 (Ab52947, Abcam), NQO1 (Ab28947, Abcam), IL‐1β (AF5103, Affinity), IL‐6 (AF6087, Affinity), Ki67 (AF0198, Affinity), CD31 (AF6191, Affinity), and collagen I (AF7001, Affinity).
6
Osteogenic Protein Expression Analysis
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The expression of osteogenesis-related protein OPN and Col-I was detected by immunofluorescence staining.(Yao et al., 2019b (link)). The BMSCs were cultured on the various microcarriers for 14 days, fixed with 4% PFA for 20 min at room temperature and then washed with PBS. The fixed cells were permeabilized with 0.25% Triton X-100 in PBS for 10 min and blocked with 5% bovine serum albumin (BSA) in PBST (0.25% Tween-20 in PBS). Next, primary anti-Col-I (1:200, AF7001, Affinity, United States) or anti-OPN (1:200, AF0227, Affinity, United States) was incubated overnight at 4°C on a shaking table. After being washed three times with PBS, the cells were exposed to the secondary antibody, goat anti-rabbit IgG (1:1,000, ab150077 Alexa Fluor®488, Abcam, United Kingdom) for 2 h at room temperature in dark. Finally, 4′,6-diamidino-2-phenylindole (DAPI; Sigma, United States) was used to stain the cell nuclei. Photos were taken on a laser confocal microscope (FV1000, Olympus, Tokyo, Japan).
7
Histological Analysis of Cardiac Fibrosis
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A segment of the heart tissue was excised, fixed in 4% paraformaldehyde for 24 h and embedded in paraffin. Serial 5-μm-thick sections were mounted and stained with hematoxylin and eosin (HE) and Masson's trichrome to examine the pathology of the heart. Furthermore, immune histology staining was performed for collagen I (1:200, AF7001, Affinity Biosciences, USA) and collagen III (1:200, AF0136, Affinity Biosciences, USA) in heart sections. Quantitative assessment of the fibrosis was determined based upon the extent of patchy and interstitial fibrosis. Each heart was observed, and at least 5 pictures were obtained in each region.
8
Peimine Fibrosis Inhibition Protocol
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Peimine (CAS: 23496-41-5) was prepared by Chengdu Munster Biotechnology Co., Ltd. (Chengdu, Sichuan, China). Bleomycin sulfate was obtained from Selleck (lot S1214) (Shanghai, China). Pirfenidone (PFD) capsules were obtained from the Beijing Kangdini Pharmaceutical Co. Ltd. (lot 150603) (Beijing, China). Antibodies against Collagen I, Collagen III, and CD68 were purchased from Affinity (AF7001, AF0136, DF7518), while antibodies against Arg-1, CD206, CTGF, TGF-β1, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were obtained from Proteintech (16001-1-AP, 60143-1-Ig, 23936-1-AP, 21898-1-AP, 60004-1-Ig). P-STAT6, STAT6, p-Akt (s473), Akt, p-p38 MAPK, p38 MAPK antibodies were obtained from Cell Signaling Technology (5397S, 56554S, 4060S, 4685S, 4511S, 8690S). FITC and Cy3-conjugated affinipure goat antirabbit IgG (H+L) were purchased from Proteintech (SA00003-2, SA00009-2).
9
Immunohistochemical Analysis of Collagen-I
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The treated rats were sacrificed and the Achilles tendon tissue (~0.5 cm) around the injured area was harvested. For IHC analysis, the paraffin-embedded sections (5 µm)
were deparaffinized by xylene, treated with a graded series of ethanol. Antigen retrieval for collagen-I was performed through incubation with Proteinase K (Servicebio) antigen retrieval
solution for 15 min at room temperature. After washing and blocking, the sections were incubated with anti-collagen-I (AF7001, Affinity Biosciences, Jiangsu, China) overnight at a dilution
of 1:100. HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (31460, 1:500, ThermoFisher, Waltham, MA, USA) were added for an hour, followed by Diaminobenzidine (DAB, Fuzhou New Step
Biotechnology Development Co., Ltd., Fujian, China). Afterwards, the sections were counterstained in hematoxylin and finally examined by microscope (OLYMPUS).
were deparaffinized by xylene, treated with a graded series of ethanol. Antigen retrieval for collagen-I was performed through incubation with Proteinase K (Servicebio) antigen retrieval
solution for 15 min at room temperature. After washing and blocking, the sections were incubated with anti-collagen-I (AF7001, Affinity Biosciences, Jiangsu, China) overnight at a dilution
of 1:100. HRP-conjugated Goat Anti-Rabbit IgG secondary antibody (31460, 1:500, ThermoFisher, Waltham, MA, USA) were added for an hour, followed by Diaminobenzidine (DAB, Fuzhou New Step
Biotechnology Development Co., Ltd., Fujian, China). Afterwards, the sections were counterstained in hematoxylin and finally examined by microscope (OLYMPUS).
10
Protein Extraction and Western Blot Analysis
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RIPA buffer (Shanghai biyuntian Biotechnology Co., Ltd, China) containing 1 mM PMSF (Shanghai biyuntian Biotechnology Co., Ltd, China) was used to lyse cells on ice for 30 min, after which samples were centrifuged for 10 min at 12000 rpm at 4°C. Protein levels in the resultant supernatants were then assessed with a BCA Protein Assay Kit (Takara, Japan), and 30 ug of protein per sample was separated via 10% SDS‐PAGE and transferred onto PVDF membranes (Shanghai biyuntian Biotechnology Co., Ltd, China). Blots were blocked with 5% BSA and then incubated overnight with appropriate primary antibodies (anti‐collagen I, Affinity, AF7001, 1:500; anti‐collagen III, Affinity, 22 734‐1‐AP, 1:500; anti‐proliferating cell nuclear antigen (PCNA), Proteintech, 10 205‐2‐AP, 1:5000; anti‐α‐SMA, Affinity, AF1032, 1:500; anti‐Bip, Proteintech, 66 574‐1‐Ig; anti‐p‐IRE1α, Affinity, AF7150, 1:2000; anti‐TRAF2, Proteintech, 26 846‐1‐AP, 1:1000; anti‐NF‐κBp65, CST, 8242S, 1:1000; anti‐ GAPDH, 60004‐1‐lg, 1:100000) at 4°C. Blots were then washed thrice with TBST, incubated for 90 min with appropriate secondary antibodies at room temperature, washed three more times, and protein bands were then detected using ECL solution (Thermo, USA). ImageJ (v 1.53a, NIH, MD, USA) was then used for densitometric analyses, with GAPDH being used for normalisation.
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