Each section was incubated overnight at 4 °C with primary antibodies targeting Type I procollagen (ab64409, rat mAb, Abcam, Cambridge, UK, cross-reacts with human), Type I collagen (600–401-103, rabbit pAb, Rockland, Limerick, PA, cross-reacts with bovine and human), Type III collagen (ab6310, FH-7A, mouse mAb, Abcam, Cambridge, UK, cross-reacts with rat and human), Type V collagen (AM10159PU-N, V13F6, mouse mAb, Acris, San Diego, CA, cross-reacts with human), keratin-14 (20R-CP002, guinea pig pAb, Fitzgerald Industries International, Acton, MA) and PDGFRbeta (MAB1263, mouse mAb, R&D Systems, Minneapolis, MN). Primary antibodies were detected using Alexa488- or Alexa594-conjugated secondary antibody (Thermo Fisher Scientific). Sections were examined with an Olympus BX51 microscope (Olympus, Tokyo, Japan) and images were captured with a DP72 controller digital camera (Olympus). The staining intensity in 6 randomly selected microscopic fields was quantified by using WINROOF2015 image-analyzing software (Mitani, Fukui, Japan, https://www.mitani-visual.jp/products/image_analys_ismeasurement/winroof/ ), as described previously10 (link).
Ab64409
Manufactured by Abcam
Sourced in United Kingdom
Ab64409 is a lab equipment product manufactured by Abcam. It is a device used for the purpose of performing laboratory experiments and analyses. The core function of this product is to facilitate the execution of specific laboratory procedures.
Automatically generated - may contain errors
Lab products found in correlation
Lsm 700, by Zeiss (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Ab6310, by Abcam (1 mentions)
Dp72 controller digital camera, by Olympus (1 mentions)
Fh 7a, by Abcam (1 mentions)
Mab1263, by R&D Systems (1 mentions)
Alexa 594 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
Alexa fluor 488 anti rat igg, by Thermo Fisher Scientific (1 mentions)
Zen 2009, by Zeiss (1 mentions)
Af 379 na, by R&D Systems (1 mentions)
Mab3171 100, by R&D Systems (1 mentions)
Ab76315, by Abcam (1 mentions)
Ab21600, by Abcam (1 mentions)
Ab150077, by Abcam (1 mentions)
4 protocols using ab64409
1
Quantitative Analysis of Extracellular Matrix Proteins
2
Multifaceted Immune Profiling by Confocal Microscopy
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CD4, IL-17, IL-4, pSTAT3 (y705), CXCR3, and procollagen levels were analyzed by confocal microscopy. Paraffin-embedded sections were incubated with 10% normal goat serum for 30 min and stained with anti-CD4 (AF-379-NA; R&D Systems), anti-IL-17 (MAB3171-100; R&D Systems), anti-IL-4 (PA5-25165; Thermo Fisher Scientific), anti-pSTAT3 (y705) (ab76315; Abcam), anti-CXCR3 (SC-133087; Santa Cruz Biotechnology, Dallas, TX), and anti-procollagen (ab64409; Abcam) antibodies. Next, the samples were reacted with anti-goat IgG-FITC (SC-2024; Santa Cruz Biotechnology), anti-mouse-IgG1-Alexa Fluor 555 (A21127; Invitrogen, Waltham, MA), anti-rabbit-IgG-PE (4050-09; Southern Biotech, Birmingham, AL), anti-rabbit-IgG APC (A-10931; Invitrogen), anti-mouse-IgG-Alexa Fluor 647 (1031-31; Southern Biotech), and anti-rat IgG-Alexa Fluor 488 (A-11006; Invitrogen)-conjugated secondary antibodies. Nuclei were stained with DAPI (D3571; Thermo Fisher Scientific). The sections were visualized using a confocal microscope (LSM 700; Carl Zeiss, Oberkochen, Germany); the colocalization area and intensity were analyzed using ZEN 2009 software (Carl Zeiss).
3
Immunofluorescent Detection of Procollagen and Elastin
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Five-micron skin sections were stained with rat anti-procollagen type I (#ab64409, Abcam, Cambridge, UK) and rabbit anti-tropoelastin (#ab21600, Abcam, Cambridge, UK) antibodies overnight at 4 °C. Slides were washed and then incubated with secondary antibodies (goat anti-rat Alexa 546 [#A-11081, Invitrogen, Carlsbad, CA] or goat anti-rabbit Alexa 488 [#ab150077, Abcam, Cambridge, UK]) for 1 h at room temperature and counterstaining of cell nuclei was performed using DAPI. For all experimental conditions, a control incubated without primary antibodies was included.
4
Quantifying Procollagen I in Skin
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Twelve skin sections for each treatment were immunostained using procollagen I antibody (ab64409, Abcam, Cambridge, MA). The amount of procollagen I was evaluated based on the intensity and distribution of immunostained procollagen I in the area corresponding to the papillary dermis, just below the epidermis. As above, 8-bit grey-scale photomicrographs were captured and transformed from RGB to the standard CIE L*a*b* color space. The L* value for each pixel in the ROI was evaluated using ImageJ software (National Institutes of Health, Bethesda, MD) with the Color Inspector 3D plug-in. Scores reflecting the amount of procollagen I detected were assigned using an image analysis algorithm proprietary to Cutech Srl (Fig 1 ).
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