Lysates were generated by incubating cells or ground tumors in RIPA buffer (Millipore). 20–30 μg of cleared lysate were analyzed by SDS-PAGE as previously described 11 (link). The following primary antibodies were used: Vinculin (1:5,000 dilution; Sigma, V9131), β-Actin (1:5,000; Sigma, A5441), Collagen I (1:500; Abcam, ab21286), Fibronectin (1:1,000; Abcam, ab2413), Smad4 (1:200; Santa Cruz, sc-7966), Smad2 p-S465/467 (1:1,000; Cell Signaling, 3108S), Smad2/3 (1:1,000; Cell Signaling, 3102S), GCN2 p-T899 (1:1,000; Abcam, ab75836), GCN2 (1:1,000; Cell Signaling, 3302S), ATF4 (1:200; Santa Cruz, sc-200), PC (1:1,000; Novus, NBP1–49536), S6K (1:1,000; Cell Signaling, 2708S), GLUL (1:1,000; Sigma, G2781), SMA (1:1,000; Millipore, CBL171). The following secondary antibodies were used: anti-rabbit HRP (1:5,000; GE, NA934V), anti-mouse HRP (1:5,000; GE, NA931). Quantification of band intensities of Collagen I relative to β-Actin or Vinculin in tumor allograft experiments was performed with Image Lab software v6.0 (Bio-Rad).
Collagen 1
Manufactured by Bio-Rad
Sourced in United States
Collagen I is a type of collagen protein produced by Bio-Rad for use in research and laboratory applications. It serves as a key structural component in various tissues and is essential for cell attachment and proliferation. The product is available in various formats to meet the diverse needs of researchers.
Automatically generated - may contain errors
Lab products found in correlation
Fibronectin, by Abcam (3 mentions)
Vinculin, by Bio-Rad (2 mentions)
Vinculin, by Merck Group (2 mentions)
Anti mouse hrp, by GE Healthcare (2 mentions)
Smad2 3, by Cell Signaling Technology (2 mentions)
Smad2 p s465 467, by Cell Signaling Technology (2 mentions)
Gcn2 p t899, by Abcam (2 mentions)
Nbp1 49536, by Cell Signaling Technology (2 mentions)
Anti rabbit hrp, by GE Healthcare (2 mentions)
Image lab software v 6, by Bio-Rad (2 mentions)
Ripa buffer, by Merck Group (2 mentions)
Smad4, by Santa Cruz Biotechnology (2 mentions)
Collagen 1, by Abcam (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Bio-Rad (2 mentions)
Axiocam hrc, by Zeiss (1 mentions)
Immunoblock, by Sumitomo Pharma (1 mentions)
Mmp 9, by Abcam (1 mentions)
F4 80, by BioLegend (1 mentions)
Cellsens software, by Olympus (1 mentions)
5 protocols using collagen 1
1
Western Blot Analysis of Tumor Proteins
2
Western Blot Analysis of Tumor Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Lysates were generated by incubating cells or ground tumors in RIPA buffer (Millipore). 20–30 μg of cleared lysate were analyzed by SDS-PAGE as previously described 11 (link). The following primary antibodies were used: Vinculin (1:5,000 dilution; Sigma, V9131), β-Actin (1:5,000; Sigma, A5441), Collagen I (1:500; Abcam, ab21286), Fibronectin (1:1,000; Abcam, ab2413), Smad4 (1:200; Santa Cruz, sc-7966), Smad2 p-S465/467 (1:1,000; Cell Signaling, 3108S), Smad2/3 (1:1,000; Cell Signaling, 3102S), GCN2 p-T899 (1:1,000; Abcam, ab75836), GCN2 (1:1,000; Cell Signaling, 3302S), ATF4 (1:200; Santa Cruz, sc-200), PC (1:1,000; Novus, NBP1–49536), S6K (1:1,000; Cell Signaling, 2708S), GLUL (1:1,000; Sigma, G2781), SMA (1:1,000; Millipore, CBL171). The following secondary antibodies were used: anti-rabbit HRP (1:5,000; GE, NA934V), anti-mouse HRP (1:5,000; GE, NA931). Quantification of band intensities of Collagen I relative to β-Actin or Vinculin in tumor allograft experiments was performed with Image Lab software v6.0 (Bio-Rad).
3
Histological Analysis of Liver Tissue
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Liver cryosections (8 μm) were mounted on glass slides, and hematoxylin and eosin and sirius red staining were performed as described.15 Immunostaining was performed by cold acetone fixation. The fixed sections were incubated with ImmunoBlock (DS Pharma Biomedical) and then incubated with Ly6G (BioLegend) or F4/80 (BioLegend) and collagen I (Bio‐Rad, Hercules, CA) or Mmp9 (Abcam) antibodies, followed by Alexa555‐labeled anti‐rat and Alexa488‐labeled anti‐rabbit secondary antibodies (Molecular Probes). Images were captured using Observer Z1 with an AxioCam HRc (Zeiss, Oberkochen, Germany) except for quantification of sirius red‐positive areas, which were analyzed using cellSens software (Olympus, Tokyo, Japan). Ly6G‐positive (+) or F4/80+ cell numbers were counted using Image J software (for Figs. 2 , 3 , 4 , 5 ). Ly6G and Mmp9 double‐positive cell numbers were counted using the BZ‐X700 hybrid cell count software (Keyence, Osaka, Japan).
4
Histological Characterization of Cell Sheets
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
After detachment cell sheets were frozen in Tissue-Tek (Sakura Finetek, USA) and 7 μm sections were obtained. Cross sections were formalin-fixed and stained by primary antibodies against Connexin43 (cat#71-0700, ThermoFisher Scientific, USA), Ki-67 (cat#ab16667, Abcam, USA), and Gata 4 (cat#PA5-29663, Thermo Fisher Scientific, USA) cleaved caspase-3 (cat#9664S, Cell signaling, USA) for 1 hour. To visualize ECM components cell sheet sections were incubated with a polyclonal antibodies specifically reactive with collagen I (cat#2150-1908, Bio-Rad, USA), collagen 3 (cat#2150-1948, Bio-Rad, USA), and the fibronectin (cat#6328-250, Abcam, USA). After washing the slides were stained for 40 min by the corresponding secondary antibodies (cat#A11001, A11008, A21203, or A21207, all Invitrogen, USA, 1:800, 1h at 37°C).
5
Immunofluorescence Staining of Cultured Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cultured cells were fixed with 10% formaldehyde for 20 min at 4 °C, then incubated overnight at 4 °C with the specific primary antibodies. They were subsequently incubated with appropriate fluorescent secondary antibodies and examined under a fluorescence microscope (BZ-X810, Keyence). The primary and secondary antibodies used were as follows: Collagen I (Bio-Rad 2150-1410, 1:300), α-SMA (Sigma A2547, 1:500), Alexa Fluor 647 Phalloidin (Thermo fisher A22287, 1:300), Alexa Fluor 488-conjugated rabbit IgG (Thermo fisher A21206, 1:400) and Alexa Fluor 555-conjugated mouse IgG (Thermo fisher A31570, 1:400).
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!