The cross sections of cell aggregates were prepared according to the procedure reported in our previous study [15] (link). The sections were then incubated overnight at 4 °C with primary antibody against collagen type I (Abcam, Cambridge, MA, UK) diluted in PBS with 10% Block Ace (DS Pharma Biomedical, Osaka, Japan). After washing with Tris-buffered saline and incubation in PBS containing 10% Block Ace and Alexa Fluor 594-conjugated secondary antibody (Thermo Fisher Scientific, Waltham, MA, USA) for 60 min, cell nuclei were stained with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific, Waltham, MA, USA) for 20 min. The sections were then washed with PBS and observed with a confocal laser scanning microscope (FV-1000; Olympus, Tokyo, Japan).
Primary antibody against type 1 collagen
Manufactured by Abcam
Sourced in United Kingdom
This primary antibody is specific for type I collagen, a major structural protein found in the extracellular matrix of various tissues. It can be used to detect and quantify type I collagen in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Total h3, by Abcam (2 mentions)
Pvdf membrane, by Thermo Fisher Scientific (2 mentions)
Chemidoc touch imaging system, by Bio-Rad (2 mentions)
Gapdh, by Merck Group (2 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Bis tris gel, by Thermo Fisher Scientific (2 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Rabbit ant chicken, by Merck Group (2 mentions)
H3k27me3, by Cell Signaling Technology (2 mentions)
Block ace, by Sumitomo Pharma (1 mentions)
Fv1000, by Olympus (1 mentions)
Alexa fluor 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Block ace, by Thermo Fisher Scientific (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Alexa fluor 488 anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Axio scope a1, by Zeiss (1 mentions)
Eclipse ti, by Zeiss (1 mentions)
Triton x 100, by Beyotime (1 mentions)
Second antibody, by Abcam (1 mentions)
6 protocols using primary antibody against type 1 collagen
1
Immunofluorescence Analysis of Collagen I
2
Collagen Type I Immunostaining in Pancreatic Tumors
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Pancreases from Rip1-Tag2 mice and Rip1-Tag2;P-sel-/- mice were fixed in 4% paraformaldehyde overnight and then embedded in optimum cutting temperature compound (OCT) and sectioned. For immunofluorescence staining, the sections were rehydrated in distilled water, blocked with 10% bovine serum albumin (BSA) and incubated with a primary antibody against collagen type I (Abcam, Cambridge, CB, UK) overnight at 4 °C. The next day, DyLight 488 or 555-conjugated anti-GPIbβ antibodies (Invitrogen, Carlsbad, CA, USA) were added to the sections, which were counterstained with 4'-6-diamidino-2-phenylindole (DAPI). For immunofluorescence staining quantitation, we randomly chose fields from at least five tumors per mouse (typically 10-15) in five to eight mice per group using a 40 × objective lens. The slides were collected, and the images were quantified using Image-Pro Plus software (IPP, version 6.0, Media Cybernetics).
3
Immunoblotting of Cellular Proteins
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Immunoblotting was performed on tissues that were lysed using ice-cold RIPA buffer supplemented with Halt Phosphatase and Protease Inhibitor Cocktail (Thermo Fisher Scientific). The samples were spun at 3,000g to remove debris and 10 μg of sample was resolved on a 4–12% Bis-Tris gel (Thermo Fisher) using SDS-PAGE. Samples were transferred onto a PVDF membrane (Thermo Fisher) and blocked in 5% milk in PBS with 0.1% Tween 20 (PBS-T). Membranes were washed 3 times in PBS-T after blocking and between each subsequent step. Membranes were incubated overnight at 4°C with primary antibody against type I collagen (Abcam, 1:1000), GAPDH (EMD Millipore, 1:1000), H3K27me3 (Cell Signaling Technology, 1:000), or total H3 (Abcam, 1:000) diluted in 1% Bovine Serum Albumin in PBS-T with 0.05% sodium azide. Subsequently, membranes were incubated with species-specific HRP-conjugated antibodies (goat anti-rabbit (Abcam); rabbit ant-chicken (EMD Millipore)), developed with Clarity Western ECL Substrate (Bio-Rad), and visualized with a ChemiDoc Touch Imaging System (Bio-Rad).
4
Immunoblotting of Cellular Proteins
5
Collagen Localization in DPC Sheets
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DPC sheets were washed twice
with PBS and fixed in 10% formalin. Then, the cell sheets were processed
for standard paraffin embedding and sectioned at a 5 μm thickness.
Sections were stained with hematoxylin and eosin (H&E), or immunohistochemical
staining was performed using a primary antibody against type I collagen
(Abcam) and secondary antibody Alexa Fluor 488 anti-rabbit IgG (ThermoFisher).
DAPI staining was used to visualize nuclei. Images were captured using
a Nikon ECLIPSE Ti, ZEISS Scope.A1 AXIO or Nikon TE 2000 microscopes
and processed using the ImageJ software.
with PBS and fixed in 10% formalin. Then, the cell sheets were processed
for standard paraffin embedding and sectioned at a 5 μm thickness.
Sections were stained with hematoxylin and eosin (H&E), or immunohistochemical
staining was performed using a primary antibody against type I collagen
(Abcam) and secondary antibody Alexa Fluor 488 anti-rabbit IgG (ThermoFisher).
DAPI staining was used to visualize nuclei. Images were captured using
a Nikon ECLIPSE Ti, ZEISS Scope.A1 AXIO or Nikon TE 2000 microscopes
and processed using the ImageJ software.
6
Immunofluorescence Assay for Type I Collagen
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Osteoblasts were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton X-100 (Beyotime Biotechnology, Beijing, China) for 5 min at room temperature. Osteoblasts were blocked with goat serum for 1 h, and incubated in primary antibody against type I collagen (Abcam, Cambridge, MA, USA; 1:200) at 4 °C overnight. Followed by PBS washing, incubation with second antibody (Abcam; 1:200) was conducted for 1 h at room temperature. The nuclei of cells were counterstained by DAPI solution (Solarbio) and images were captured using fluorescence microscopy (Nikon, Tokyo, Japan).
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