Dissected tissue was homogenized by manual grinding in cold RIPA buffer containing protease and phosphatase inhibitor cocktails (Thermo Scientific) and molecular grinding resin (G Biosciences). Lysates were subjected to three freeze/thaw cycles and clarified by centrifugation at 12,000 × g for 30 min at 4 °C. Protein concentration was determined with a protein assay kit (Micro BCA, Thermo Scientific). Protein lysates (3–5 μg) were separated by polyacrylamide SDS-PAGE gels [15% polyacrylamide for LC3 detection (NB100-2331 from Novus Biologicals) and 7% polyacrylamide for fibronectin (SC-8422 from Santa Cruz) and SQSTM1 (P0067 from MilliporeSigma)] and transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% nonfat dry milk in 0.1% Tween-20/TBS and incubated overnight with primary antibodies. The bands were detected by incubation with a secondary antibody conjugated to horseradish peroxidase and chemiluminescence substrate (ECL; GE Healthcare and ECL2, Thermo Scientific). Blots were scanned and analyzed by densitometry using Image J. β-Actin (sc-69879) was used for loading control.
Sc 8422
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-8422 is a laboratory equipment product from Santa Cruz Biotechnology. It is designed for general laboratory use.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Merck Group (3 mentions)
Mab374, by Merck Group (3 mentions)
A5228, by Merck Group (3 mentions)
Perioxidase conjugated secondary antibodies, by Jackson ImmunoResearch (2 mentions)
N cadherin, by Santa Cruz Biotechnology (2 mentions)
Sc 293182, by Santa Cruz Biotechnology (2 mentions)
Ab52903, by Abcam (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Microbca, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Molecular grinding resin, by G Biosciences (1 mentions)
Nb100 2331, by Novus Biologicals (1 mentions)
P0067, by Merck Group (1 mentions)
Protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Axio imager z1 microscope, by Zeiss (1 mentions)
Lsm 510, by Zeiss (1 mentions)
Objective lens, by Zeiss (1 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
C2456, by Merck Group (1 mentions)
Sc 53015, by Santa Cruz Biotechnology (1 mentions)
17 protocols using sc 8422
1
Western Blot Analysis of Autophagy Markers
2
Immunofluorescent Visualization of ECM Components
3
Evaluating Protein Levels in Pancreatic Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blotting was performed to evaluate the protein expression levels of α-SMA and fibronectin. Pancreatic tissue homogenates (120 μg protein/lane) were separated by 8% to 12% SDS-PAGE and transferred onto nitrocellulose membranes (Amersham Inc., Arlington Heights, IL, USA) by electroblotting. Membranes were then incubated with antibodies against α-SMA (1:5,000, sc-53015; Santa Cruz Biotechnology, Dallas, TX, USA), fibronectin (1:100, sc-8422; Santa Cruz Biotechnology), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:3,000, sc-47724; Santa Cruz Biotechnology) at 4°C overnight. Following incubation, membranes were washed with TBS with Tween-20. The primary antibodies were then detected using horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized using enhanced chemiluminescence detection system (Santa Cruz Biotechnology) according to the manufacturer’s instruction. GAPDH was used as loading control. Expression levels were standardized to GAPDH expression level. Further, data were processed and quantified using volume analysis and molecular analysis software, respectively.
4
ECM-Coated Polymeric Mesh Scaffolds
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After the decellularization, CDM was harvested by gentle pipetting, transferred to 50 mL tubes, and vigorously agitated using a homogenizer (HG-3000, SMT, Japan) until a homogeneous aqueous phase was formed. The polymer mesh scaffolds were then immersed into the CDM suspension solution with a mild agitation and incubated for 24 h. The CDM-coated mesh scaffolds were then freeze-dried overnight. Fibronectin (FN; BD Biosciences)-coated mesh scaffolds were also prepared by soaking the scaffolds in FN solution (50 μg/mL in distilled water) at 37 °C for 1 h. The FN-coated scaffolds were then rinsed with distilled water and freeze-dried. The surface morphology of the FN- and CDM-coated mesh scaffolds was observed via scanning electron microscopy (SEM; Phenom G2 Pro Desktop). In addition, the distribution of the CDM in the mesh scaffolds was visualized via immunofluorescence staining of fibronectin using mouse monoclonal antibody (SC-8422; Santa Cruz Biotechnology) and Alex Fluor 488-conjugated secondary antibody (goat anti-mouse IgG; Invitrogen), respectively.
5
Immunofluorescence Analysis of ECM Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunofluorescence (IF) staining of fibronectin (FN), collagen type I alpha 1 (COL1A1), and F‐actin was performed as previously reported.
29 (link) Briefly, the samples were fixed with 4% formaldehyde solution for 15 min and rinsed with PBS. After that, the samples were penetrated with 0.3% TritonX‐100 in PBS for 10 min and rinsed with PBS, followed by blocking incubation (5% normal goat serum in PBS) for 1 h at room temperature. Primary antibodies were diluted in 1% BSA/PBS as below: FN (SC‐8422, Santa Cruz Biotechnology) (1:50), COL1A1 (ab21286, Abcam) (1:200), and F‐actin (FITC‐phalloidin) (CA1620, Solarbio) (1:100). After incubation with primary antibodies at 4°C overnight and PBS washing, secondary antibodies conjugated with Alexa Fluor® 555 or Alexa Fluor® 488 (Cell Signaling Technology) were applied except F‐actin stained samples. Imaging was caught under an inverted fluorescence microscope.
29 (link) Briefly, the samples were fixed with 4% formaldehyde solution for 15 min and rinsed with PBS. After that, the samples were penetrated with 0.3% TritonX‐100 in PBS for 10 min and rinsed with PBS, followed by blocking incubation (5% normal goat serum in PBS) for 1 h at room temperature. Primary antibodies were diluted in 1% BSA/PBS as below: FN (SC‐8422, Santa Cruz Biotechnology) (1:50), COL1A1 (ab21286, Abcam) (1:200), and F‐actin (FITC‐phalloidin) (CA1620, Solarbio) (1:100). After incubation with primary antibodies at 4°C overnight and PBS washing, secondary antibodies conjugated with Alexa Fluor® 555 or Alexa Fluor® 488 (Cell Signaling Technology) were applied except F‐actin stained samples. Imaging was caught under an inverted fluorescence microscope.
6
Renal Expression Analysis of Key Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Renal expression of nephrin (1:1,000, sc-377246), fibronectin (1:1,000, sc-8422), type IV collagen (1:1,000, sc-11360), ACE (1:1,000, sc-23909), NOX4 (1:1,000, sc-30141), MCP-1 (1:2,000, sc-28879) (all from Santa Cruz Biotech, Santa Cruz, CA, USA), and AT1R (1:1,000, ab-18801) (Abcam, Cambridge, UK) was detected by Western blot analysis. Renal cortex tissues were homogenized in protein lysis buffer. All samples were centrifuged at 13,000 ×g for 20 minutes. The supernatants were mixed with the sample buffer and heated for 5 minutes at 95°C. Sample aliquots containing 15 to 30 μg of protein were loaded on sodium dodecyl sulphate polyacrylamide gel electrophoresis (8% to 12%) and subsequently transferred to polyvinylidene difluoride membranes. The membranes were incubated at 4°C overnight with primary antibodies and washed three times in Tris-buffered saline with 0.1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. The gels were visualized using an enhanced chemiluminescence gel electrophoresis system (Amersham Biosciences, Buckinghamshire, UK). The intensity of the bands was measured using ImageJ software version 1.50i (NIH, Bethesda, MD, USA).
7
Western Blot Analysis of Fibroblast Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HGFs were washed twice with 4 °C PBS and protein isolated in a RIPA buffer (Sigma-Aldrich, Oakville, ON, Canada) containing protease (Roche Diagnostics GmbH, Mannheim, Germany) and phosphatase inhibitor (Calbiocam, Billerica, MA, USA) cocktails. Protein concentration was determined by Pierce® BCA Protein assay kit (Pierce, Waltham, MA, USA). An amount of 25 μg proteins of each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and blocked with 5% dried milk in TBS-T. Primary antibodies for fibronectin (sc-8422; Santa Cruz Biotechnology; 1:1000, Dallas, TX, USA), α-SMA (A5228; Sigma-Aldrich, 1:1000, Oakville, ON, Canada) and GAPDH (MAB374; Millipore; 1:2000, Toronto, ON, Canada) were used. Detection was performed with appropriate perioxidase-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA; 1:2000), which were developed with SuperSignal Western Pico Chemiluminescence Substrate (Pierce, Waltham, MA, USA).
8
Sulforaphane-Mediated Regulation of NRF2 Pathway
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Sulforaphane (S6317), as well as anti-mouse IgG (HRP) (1:3000, A-9044) and anti-rabbit IgG (HRP) (1:3000, A-0545) antibodies were purchased from Sigma-Aldrich. LY294002 (ab120243) was purchased from Abcam. The rabbit normal IgG and antibodies against NRF2 (1:1000, sc-13032), sMAF (1:1000, sc-22831), NQO1 (1:1000, sc-32793), FN1 (1:1000, sc-8422), COL1A1 (1:1000, sc-293,182), α-SMA (1:1000, sc-53142), N-Cadherin (1:500, sc-7939) and GAPDH (1:3000, sc-32233) were purchased from Santa Cruz Biotechnology. The antibody against E-Cadherin (1:1000, 610,181) was purchased from BD Biosciences. Antibodies against AKT (1:2000, 4691S) and phospho-AKT (Ser473) (1:1000, 4060S) were purchased from Cell Signaling Technology. TGF-β (100–21) was purchased from PeproTech.
9
Western Blot Analysis of Hepatic Fibrosis Markers
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver tissues and HSC-T6 cells were lysed with RIPA buffer (#ab156034, Abcam, Cambridge, UK) containing a phosphatase inhibitor and PMSF (#ab141032, Abcam, Cambridge, UK). Protein samples were denatured and separated by 10–15% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Burlington, MA, USA). The membranes were blocked and incubated with primary antibodies. The primary antibodies were as follows: collagen I (1:1000; #ab270993, Abcam, UK), TGF-β1 (1:500; #sc-130348, Santa Cruz, CA, USA), collagen III (1:1000; #ab184993, Abcam, UK), Smad2 (1:500; #sc-393312, Santa Cruz, CA, USA), Smad3 (1:500; #sc-101154, Santa Cruz, CA, USA), p-Smad2 (1:1000; #ab280888, Abcam, Cambridge, UK), p-Smad3 (1:1000; #ab52903, Cambridge, Abcam, UK), LC3 (1:1000; #ab192890, Abcam, Cambridge, UK), Beclin-1 (1:1000; #ab207612, Abcam, Cambridge, UK), P62 (1:1000; #sc-28359, Santa Cruz, CA, USA), α-SMA (1:1000; #55135-1-AP Proteintech, Rosemont, IL, USA), fibronectin (FN) (1:500; #sc-8422, Santa Cruz, CA, USA), and GAPDH (1:1000; #ab8245, Abcam, Cambridge, UK). Subsequently, the membranes were incubated with HRP anti-rabbit IgG (1:2000; #ab288151, Abcam, Cambridge, UK). Finally, the bands were visualized using the ECL- chemiluminescent kit (Proteintech, Rosemont, IL, USA) and quantified using ImageJ software.
10
Western Blot Analysis of HGFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HGFs were cultured on collagen type I and rhPN pre-coated plates as previously described for 1 and 7 days. Western blotting was performed as previously described68 (link),69 (link). In brief, proteins were harvested with RIPA buffer (Sigma Aldrich) containing protease and phosphatase inhibitor cocktails. Protein concentration was determined by Pierce® BCA Protein assay kit (Pierce; Waltham, MA). 25 μg proteins of each sample were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. Membranes were washed with Tris-buffered saline containing 0.05% Tween-20 (TBS-T) and blocked with 5% dried milk in TBS-T. Primary antibodies for fibronectin (sc-8422; Santa Cruz Biotechnology; 1:1000), α-SMA (A5228, Sigma Aldrich, 1:1000), and GAPDH (MAB374; Millipore; 1:2000) were used to incubate the membranes for 12 hours. Detection was with appropriate peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch; West Grove, PA; 1:2000), which were developed with Clarity Western ECL substrate (Bio-Rad; Hercules, CA). Densitometry analysis was performed using ImageJ software version 10.2 (National Institutes of Health; Bethesda, MD).
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!