The protein levels were measured by Western immunoblot in cells solubilized in RIPA buffer (100 μL) containing a cOmplete™ protease inhibitor cocktail (Roche, Rotkreuz, Switzerland), as explained earlier [11 (link)]. Anti-collagen VI antibody (catalog number ab6588, Abcam, Cambridge, UK) and anti-rabbit IgG for the secondary antibody (catalog number S9169, Sigma®) were diluted at 1:500 and 1:10,000, respectively. Quantification of protein was undertaken by normalizing the size of the band of protein of interest against the total protein load quantified using the stain-free technique and ImageLabSoftware V4.1 (Bio-Rad, Hercules, CA, USA) [20 (link),21 (link)].
Anti collagen 6 antibody
Manufactured by Abcam
Sourced in United Kingdom
Anti-collagen VI antibody is a laboratory reagent used to detect and measure the presence of collagen VI, a structural protein found in the extracellular matrix of various tissues. This antibody can be utilized in techniques such as Western blotting, immunohistochemistry, and ELISA to analyze collagen VI expression and distribution.
Automatically generated - may contain errors
Lab products found in correlation
Image lab software v 4, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Alexa fluor 568 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Autostainer plus, by Agilent Technologies (1 mentions)
Tcs sp8, by Leica (1 mentions)
Hoechst 33258, by Thermo Fisher Scientific (1 mentions)
Alexa fluor plus 555, by Thermo Fisher Scientific (1 mentions)
4 protocols using anti collagen 6 antibody
1
Quantifying Collagen VI Protein Levels
2
Immunofluorescence Staining of Adipose Tissue
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Adipose tissue was prepared for immunofluorescence staining in the following manner. Formalin-fixed, paraffin-embedded adipose tissue samples were cut into 5 μm sections, deparaffinized in xylene, and rehydrated through a series of decreasing concentrations of ethanol. The sections were pretreated with Dako Cytomation Target Retrieval Solution pH 9.0 buffer (Cat# S236884-2). The sections were incubated with rabbit polyclonal anti-collagen VI antibody (Abcam, Cat# 6588, dilution 1:250 (v/v)) for 1 h at room temperature. After incubation with primary antibodies, the sections were repeatedly rinsed with PBS and incubated for 1 h at room temperature with Alexa Fluor 568 donkey anti-rabbit IgG (Molecular Probes, Cat# A10042, dilution 1:250 (v/v)) as the secondary antibody. All the antibodies were diluted in Dako Ready-to-use Antibody Diluent (Cat# S080981-2). The incubation steps were carried out with Dako Auto Stainer Plus. Negative control slides were run in parallel with positive slides by applying rabbit IgG isotype control antibody (Cat# 02-6120) diluted at the same protein concentration as the antibody. The sections were sealed with ProLong Gold Antifade Mountant (Cat# 36935) with 4' 6-diamidino-2-phenylindole (DAPI). Subsequent scanning, imaging, and analysis of the slides were performed as previously described.
3
Collagen VI Expression in Tumor Tissue
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Paraffin-embedded tumor tissue slides were labeled with an anti-collagen VI antibody (1:250, Abcam, cat# ab182744). Next, the slides were incubated with a goat anti-rabbit IgG antibody labeled with HRP (Shanghai Gene Company, GK500705) for 40 min, stained with DAB substrate for 2–10 min and counterstained with hematoxylin.
4
Immunofluorescence Staining of Tumor Cells
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Immunofluorescence staining was used to analyze 4% paraformaldehyde-fixed, 0.1% Triton X-100-permeabilized WT MCA205 and Six1−/− MCA205 cells labeled with an anti-collagen VI antibody (1:200, Abcam, cat# ab182744) at 4 °C overnight. After washing three times, the cells were incubated with an Alexa Fluor Plus 555-conjugated (1:500, Invitrogen, A32732) secondary antibody for 1 h. Cell nuclei were counterstained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 min at RT. Images were acquired using a confocal microscope (Leica TCS SP8).
Tumors were harvested on day 8 or 12, fixed in 4% paraformaldehyde for 24 h, dehydrated in a 30% (wt/vol) sucrose solution for 48 h and finally embedded in OCT at room temperature. Four-micrometer frozen sections of tumor tissues were obtained and fixed in ice-cold acetone for 15 min. After blocking with 5% goat serum in PBS for 1 h, the tumor tissue sections were incubated with primary antibodies against CD8a (1:100, Abcam, ab217344) at 4 °C overnight. After washing three times, the tumor tissue sections were incubated with an Alexa Fluor Plus 555-conjugated (1:500, Invitrogen, A32732) secondary antibody for 1 h. Cell nuclei were counterstained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 min at RT. Images were acquired using a confocal microscope (Leica TCS SP8) and analyzed with ImageJ software.
Tumors were harvested on day 8 or 12, fixed in 4% paraformaldehyde for 24 h, dehydrated in a 30% (wt/vol) sucrose solution for 48 h and finally embedded in OCT at room temperature. Four-micrometer frozen sections of tumor tissues were obtained and fixed in ice-cold acetone for 15 min. After blocking with 5% goat serum in PBS for 1 h, the tumor tissue sections were incubated with primary antibodies against CD8a (1:100, Abcam, ab217344) at 4 °C overnight. After washing three times, the tumor tissue sections were incubated with an Alexa Fluor Plus 555-conjugated (1:500, Invitrogen, A32732) secondary antibody for 1 h. Cell nuclei were counterstained with Hoechst 33258 (Thermo Fisher Scientific, H3569) for 5 min at RT. Images were acquired using a confocal microscope (Leica TCS SP8) and analyzed with ImageJ software.
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