hTERT-RPE1 cells (American Type Culture Collection; CRL-4000) were seeded (2 × 105 cells per well in 6-well plates). Each stealth RNA (100 pmol) of Opti-Mem medium (Life Technologies) was transfected using Lipofectamine RNAi Max (Life Technologies) followed by incubation for 24h at 37°C. Trypsin treatment was used to collect RNAi-transfected cells from wells which were resuspended in 2 ml of 10% FBS (Hyclone, ThermoFisher Scientific)-DMEM. These resuspensions were seeded to 6 wells of a 12-well plate (Hydrocell, CellSeed Japan) and incubated for 48 hr at 37°C. Spheroids were fixed in 3% formalin and subjected to immunostaining. Reagents used for immunostaining: anti-β-catenin (BD transduction, 610154, 1:200), anti-FN (Sigma F3648, 1:500), Alexa Fluor 546 Phalloidin (Invitrogen, A22283,1:200). For the list of primers see Supplementary Table 5 .
Anti fn
Manufactured by Merck Group
Sourced in United States
Anti-FN is a laboratory equipment product designed for the detection and measurement of fibronectin (FN) in biological samples. It functions as a key tool for researchers and scientists in various fields of study.
Automatically generated - may contain errors
Lab products found in correlation
Anti α sma, by Merck Group (5 mentions)
Anti f4 80, by Thermo Fisher Scientific (3 mentions)
Rhodamine phalloidin, by Thermo Fisher Scientific (3 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Anti β catenin, by BD (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 546 phalloidin, by Thermo Fisher Scientific (2 mentions)
Htert rpe1 cells, by American Type Culture Collection (2 mentions)
Anti collagen 1, by Southern Biotech (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Anti gapdh, by Santa Cruz Biotechnology (2 mentions)
Phosphatase inhibitor cocktail 1 and 2, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti itgb2, by Abcam (2 mentions)
Eclipse 80i epifluorescence microscope, by Nikon (2 mentions)
Anti α sma, by Abcam (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
17 protocols using anti fn
1
Generating 3D Spheroid Culture from hTERT-RPE1 Cells
2
Immortalized MEF Culture and FN Matrix Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immortalized MEFs (provided by S. Minguet, University of Freiburg) were maintained as a confluent culture at 37 °C and 5% CO2 in DMEM (4.5 g liter–1 glucose; Gibco) supplemented with 10% heat-inactivated FCS, 2 mM l -glutamine, 50 U ml–1 penicillin, 50 µg ml–1 streptomycin and 50 μM β-mercaptoethanol. Stroma-derived 3D FN matrices were produced as previously described51 (link). For matrix production, MEFs were seeded at 0.5 × 105 cells per well in 8-well imaging chambers (Lab-Tek) for live-cell imaging or at 1.25 × 105 cells per well in 24-well plates containing round coverslips (12 mm in diameter) for static immunofluorescence imaging. Cells were cultured for 9 d, and fluorescent labeling of matrices was achieved by adding 1 µg ml–1 rhodamine-coupled FN (Cytoskeleton) at days 3, 5 and 7, allowing incorporation of fluorescently labeled FN into the secreted matrices. For static immunofluorescence analysis, the extracted FN matrices were stained with anti-FN (Sigma) and fluorescently labeled anti-rabbit (Thermo Fisher).
3
Immunofluorescence Staining of Wound Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
10-µm frozen tissue sections were rehydrated in 0.2% Tween 20/PBS for 10 min, fixed in 3.7% formaldehyde, permeabilized in 0.5% Triton X-100, blocked in 10% heat-inactivated goat serum/5% milk/PBS for 1 h, and then stained with anti–cleaved caspase 3, anti-CD31 (BD), anti–LN-332 (Abcam), anti-FN (Sigma-Aldrich), anti–MMP-9 (Sigma-Aldrich), or anti–keratin 14 (Covance). Immunostaining was also performed using antibodies against integrin α2 (CD49b; EMD Millipore), α5 (5H10-27; BD), and α6 (GoH3; EMD Millipore). Secondary antibodies were Alexa Fluor 488 goat anti–rat IgG, Alexa Fluor 488 goat anti–hamster IgG, Alexa Fluor 594 goat anti–mouse IgG, or Alexa Fluor 594 goat anti–rabbit IgG (Molecular Probes), as appropriate. Images were collected on an Eclipse 80i upright microscope using a Spot camera. For assessment of wound immunostaining, the field within the wound bed below the reepithelialized epidermis was imaged. For assessment of vessel density within papillomas, nonnecrotic tumor regions were imaged. CD31 and MMP-9 staining was quantified using ImageJ software.
4
Protein Expression Analysis of PTCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Proteins from cultured PTCs or kidneys or mitochondria were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. The blots were incubated with anti-SIRT3 (cat: 5490, CST), anti-Acetyl-lysine (cat: ab22550, Abcam, Cambridge, UK), anti-E-Cadherin (cat: 610181, BD Biosciences, San Jose, CA, USA), anti-Na/K-ATPase (cat: 3010, CST; ab76020, Abcam), anti-AQP1 (cat: AB2219, Millipore, Billerica, MA, USA), anti-FN (cat: F3648, Sigma Aldrich, St. Louis, MO, USA), anti-Collagen I (cat: 1310-01, Southern Biotech, Birmingham, AL, USA), anti-PDH-E1α (cat: ab110334, Abcam), anti-p-PDH (Ser293)(cat: ab92696, Abcam), anti-CPT1a (cat: ab128568, Abcam), anti-ATP5O (cat: ab110276, Abcam), anti-α-tubulin (cat: T9026, Sigma Aldrich), and anti-SDHA (cat: 11998, CST). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health Image J software package.
5
Western Blot Analysis of Protein Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed as previously described [17 (link)]. The primary antibodies used were as follows: anti-PTEN (Abcam Cat# ab32199, RRID:AB_777535; Cell Signaling Technology Cat# 9188, RRID:AB_2253290; Proteintech Cat# 60300-1-Ig, RRID:AB_2881415), anti-NGAL (Abcam Cat# ab63929, RRID:AB_1140965), anti-FN (Sigma-Aldrich Cat# F3648, RRID:AB_476976), anti-Col-I (Boster Biological Technology Cat# BA0325, RRID:AB_2891224), anti-α-SMA (Sigma-Aldrich Cat# A5228, RRID:AB_262054), anti-PCNA (Proteintech Cat# Biotin-60097, RRID:AB_2883063), anti-E-Cadherin (Cell Signaling Technology Cat# 14472, RRID:AB_2728770), anti-Flag (Proteintech Cat# 20543-1-AP, RRID:AB_11232216), anti-CHMP2A (Proteintech Cat# 10477-1-AP, RRID:AB_2079470), anti-SQSTM1/p62 (Cell Signaling Technology Cat# 39749, RRID:AB_2799160), anti-LC3B (Cell Signaling Technology Cat# 83506, RRID:AB_2800018), anti-LAMP1 (Cell Signaling Technology Cat# 15665, RRID:AB_2798750), anti-β-tubulin (Tianjin Sungene Biotech Cat# KM9003, RRID:AB_2744678), and anti-GAPDH (Tianjin Sungene Biotech Cat# KM9002, RRID:AB_2721026). Western blotting was performed at least three times, independently. Quantification was performed by measuring the signal intensity using the software ImageJ (National Institutes of Health).
6
Immunohistochemical Staining Techniques
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
H&E and Masson staining, immunohistochemical and immunofluorescence staining were performed according to an established procedure. The following primary antibodies for immunohistochemical and immunofluorescence staining were used: anti-Acetyl-lysine (cat: 9441, CST), anti-SIRT3 (cat: 2627, CST; cat: 365175, Santa Cruz), anti-Na/K-ATPase (cat: sc-28800, Santa Cruz), anti-FN (cat: F3648, Sigma Aldrich), and anti-Collagen I (cat: 1310-01, Southern Biotech).
7
Protein Expression Analysis in Kidney Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured NRK-49F cells were lysed in 1 × sodium dodecyl sulfate sample buffer. The kidneys were lysed with radio immunoprecipitation assay (PIPA) solution containing 1% NP40, 0.1%sodium dodecyl sulfate, 100 mg/ml phenylmethanesulfonylfluoride, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma, St Louis, MO) on ice. The supernatants were collected after centrifugation at 13,000 g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay (BCA Kit; Pierce Thermo-Scientific, Rockford, IL) according to the manufacturer’s instructions. Equal amount of protein was loaded into 10 or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride membranes. The primary antibodies were as follows: anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, Dallas, TX), anti-p-Akt (Ser473) (cat: 3868, Cell Signaling Technology), anti-p-Akt (Thr308) (cat: 2965, Cell Signaling Technology), anti-p-Smad3(Ser423/425) (cat: ab52903, Abcam), anti-FN (cat: F3648, Sigma-Aldrich), anti-α-SMA (cat: A5228, Sigma-Aldrich), and anti-type I collagen (cat: AB765P, Millipore). Quantification was performed by measuring the intensity of the signals with the aid of National Institutes of Health Image J software package.
8
Generating 3D Spheroid Culture from hTERT-RPE1 Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
hTERT-RPE1 cells (American Type Culture Collection; CRL-4000) were seeded (2 × 105 cells per well in 6-well plates). Each stealth RNA (100 pmol) of Opti-Mem medium (Life Technologies) was transfected using Lipofectamine RNAi Max (Life Technologies) followed by incubation for 24h at 37°C. Trypsin treatment was used to collect RNAi-transfected cells from wells which were resuspended in 2 ml of 10% FBS (Hyclone, ThermoFisher Scientific)-DMEM. These resuspensions were seeded to 6 wells of a 12-well plate (Hydrocell, CellSeed Japan) and incubated for 48 hr at 37°C. Spheroids were fixed in 3% formalin and subjected to immunostaining. Reagents used for immunostaining: anti-β-catenin (BD transduction, 610154, 1:200), anti-FN (Sigma F3648, 1:500), Alexa Fluor 546 Phalloidin (Invitrogen, A22283,1:200). For the list of primers see Supplementary Table 5 .
9
Protein Quantification and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BMDMs were lysed in 1×SDS sample buffer. Kidneys were lysed with RIPA buffer containing 1% NP-40, 0.1% SDS, 100 mg/ml PMSF, 1% protease inhibitor cocktail, and 1% phosphatase I and II inhibitor cocktail (Sigma-Aldrich) on ice. The supernatants were collected after centrifugation at 16,000 g at 4 °C for 30 min. Protein concentration was determined by bicinchoninic acid protein assay (BCA Protein Assay Kit, Pierce Thermo-Scientific, Rockford, IL) according to the manufacturer’s instruction. The primary antibodies were anti-PP2Ac (cat: 2038, Cell Signaling Technology, Boston, MA, USA, 1:1000), anti-PP2Acα (cat: ab106262, Abcam, Cambridge, UK, 1:1000), anti-PP2Acβ (cat: ab168371, Abcam, 1:1000), anti-methyl-PP2Ac (L309) (cat: ab66597, Abcam, 1:1000), anti-Itgb2 (cat: ab119830, Abcam, 1:1000), anti-Epac1 (cat: ab124162, Abcam, 1:1000), anti-FN (cat: F3648, Sigma-Aldrich, 1:10000), anti-Stat6 (cat: ab32520, Abcam, 1:1000), anti-p-Stat6 (T645) (cat: BS4186, Bioworld Technology, Nanjing, China, 1:1000), anti-Rap1a/b (cat: 4938, Cell Signaling Technology, 1:1000), anti-tubulin (cat: sc53646, Santa Cruz Biotechnology, 1:10000), and anti-GAPDH (cat: FL-335, Santa Cruz Biotechnology, 1:5000). Quantification was performed by measuring the intensity of the signals with the aid of the National Institutes of Health ImageJ software package.
10
Immunohistochemical Analysis of Kidney Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Kidney cryosections at 3-μm thickness were fixed for 15 min in 4% paraformaldehyde, followed by permeabilization with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min at room temperature. After blocking with 2% donkey serum for 60 min, the slides were immunostained with anti-FN (cat.: F3648, Sigma-Aldrich, St Louis, MO), anti-α-SMA (cat.: A5228, Sigma-Aldrich), anti-Rictor (cat.: A300-459A, Bethyl Laboratories), anti-F4/80 (cat.: 14–4801, eBioscience, San Diego, CA), anti-p-Akt (Ser473), or anti-Fsp1 (cat.: A511401, Dako, Carpinteria, CA). Cells cultured on coverslips were washed twice with cold 1 × PBS and fixed with cold methanol/acetone (1:1) for 10 min at −20 ºC. After three extensive washings with 1 × PBS, the cells were treated with 0.1% TritonX-100 for 5 min and blocked with 2% normal donkey serum in 1 × PBS buffer for 40 min at room temperature and incubated with the anti-Rictor, anti-FN, or anti-α-SMA, followed by staining with fluorescein isothiocyanate or tetramethylrhodamine-conjugated secondary antibody. Cells were also stained with 4',6-diamidino-2-phenylindole to visualize the nuclei. Slides were viewed with a Nikon Eclipse 80i Epi-fluorescence microscope equipped with a digital camera.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!