To investigate the immune response after injection of various materials such as saline, SP, and SAP-SP, macrophage markers including CD68 (mouse anti-CD68 monoclonal antibody, ab201340, Abcam) and CD206 (goat anti-CD206 polyclonal antibody, sc-34,577, Santa Cruz Biotechnology) were used for immunofluorescence analysis at a 1:100 dilution. Alexa Fluor 488 donkey anti-goat IgG and Alexa Fluor 594 rabbit anti-mouse IgG were used as the secondary antibodies at a 1:1000 dilution. DAPI was used to counterstain nuclei. The macrophage activity of the specimens was examined according to protocols used in a previous study [27 (link)].
Sc 34577
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-34577 is a protein detection reagent. It is used in laboratory procedures for the identification and analysis of specific proteins in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab955, by Abcam (2 mentions)
Ab201340, by Abcam (1 mentions)
Ab53004, by Abcam (1 mentions)
Ab96723, by Abcam (1 mentions)
Ab20346, by Abcam (1 mentions)
Opera phenix, by PerkinElmer (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Ab125212, by Abcam (1 mentions)
Sc 15368, by Santa Cruz Biotechnology (1 mentions)
Las af v 3 1 0 build 8587, by Leica Microsystems (1 mentions)
Ab5694, by Abcam (1 mentions)
Leica dm4000b microscope, by Leica Microsystems (1 mentions)
Ab6640, by Abcam (1 mentions)
Fluorescence microscope, by Leica camera (1 mentions)
6 protocols using sc 34577
1
Analyzing Macrophage Responses to Biomaterials
2
Immunohistochemical Identification of Macrophage Subtypes
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Immunohistochemical analyses were performed on the 20 paraffin-embedded tissue samples to locate the different cells. We identified macrophages with mouse anti-CD68 (dilution 1/50, EBM11 Clone, ref M0718 DAKO Corporation) antibody for the overall macrophage population and goat anti-CD206 (dilution 1/50, ref SC-34577 Santa Cruz Biotechnologies) antibody to discriminate the CD68+MR/CD206− (M1) and CD68+MR/CD206+ (M2) subtypes. Incubation of primary antibodies lasted for 2 h except for anti-CD206 which lasted for 20 h. The SMCs were identified with mouse anti-alpha-smooth muscle actin (α-SMA) (dilution 1/50, ref M0851 DAKO Corporation) antibody. Immunostaining used the appropriate biotinylated secondary antibodies (dilution 1/200, Vector laboratories), streptavidin-horseradish peroxidase (ABC kit, Vectastin), and the AEC substrate-chromogen system (Sigma) for visualization. Finally, slides were analyzed with an Axioplan 2 microscope, which included an HRc camera (AxioVision-Deconvolution 3D). Negative controls were performed by omission of the primary antibody and substitution with an unrelated primary antibody. As expected, both controls yielded negative results.
3
Quantifying M2 Macrophage Recruitment in ePTFE Grafts
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To identify M2 macrophage recruiting effect by coating, samples were double stained with CD68 (mouse, ab955, Abcam, 1:100) and CD206 (goat, sc-34577, Santa Cruz Biotechnology, 1:100). Alexa Fluor 594 rabbit anti mouse lgG (1:1000) and Alexa Fluor 488 donkey anti-goat (1:1000) were used for secondary antibodies. Also, samples were counterstained with DAPI to confirm the nuclei. After staining, the lumen and interior of ePTFE vascular grafts (samples) were observed in 5 random field at 200X magnification with a blinded rater in the border zone. The areas of positive stained samples were analyzed as the mean per unit area (µm²/ mm²) with the image J.
4
Immunofluorescence analysis of macrophage phenotypes
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The frozen samples were embedded in optimum cutting temperature solution and fixed in acetone, then, incubated with blocking buffer. Sections were incubated with primary antibodies col1a2 (Abcam, ab96723) and vimentin (Abcam, 200 ab20346) for 2 hours at room temperature, and then incubated with Alexa Fluor 488- and 555-conjugated secondary antibodies for 1 hour. Then, slides were rinsed and mounted with DAPI mounting solution. The collagen positive area and vimentin positive area were analyzed by Image-Pro plus 6.0 (Media Cybernetics, America), then, the positive areas relative to cell numbers were calculated respectively. Three field of view were selected and results were expressed by relative magnitude. To characterize macrophage phenotype, Sections were incubated with primary antibodies overnight at 4 °C. Primary antibodies against the pan-macrophage marker CD68 (Abcam, ab955), the M1 macrophage marker CD86 (Abcam, ab53004), and the M2 macrophage marker CD 206 (Santa Cruz, sc-34577) were used. Sections were washed and incubated with Alexa Fluor 488-, 555- and 647- fluorescently conjugated secondary antibodies. Nuclei were labeled with DAPI. Photographs were acquired and analyzed with an Opera Phenix (PerkinElmer Inc., UK).
5
Immunochemical Characterization of Transplanted Tissue
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A set of markers selected for immunochemistry included macrophage‐specific CD68 (ab125212, Abcam), М2 activated macrophage‐specific CD206 (sc‐34577, Santa Cruz Biotechnology), collagen type I (RAQ C11, Imtek, Russia), smooth muscle actin (αSMA) (ab5694, Abcam), skeletal muscle‐specific troponin I (sc‐15368, Santa Cruz Biotechnology). Cryosections were stained with antibodies according to recommendations of the manufacturers. Morphometric study was conducted using Leica DM 4000 B microscope with LAS AF v.3.1.0 build 8587 software (Leica Microsystems) at ×400 magnification images. Relative counts of CD68+ and CD206+ cells within the transplantation area were calculated for at least 100 fields of view per group per time‐lapse after surgery.
6
Adipose Tissue Histological Analysis
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Samples from iBAT, iWAT and eWAT were fixed for 24 hours in 4% paraformaldehyde and embedded in paraffin. Paraffin blocks were cut into 5 mM sections and stained with hematoxylin and eosin (H&E) or used for immunohistofluorescence (IHF) staining. For IHF, rehydrated tissue sections were blocked with 3% BSA for 1h at room temperature. Preparations were then incubated with rat antimouse F4/80 (ab6640, Abcam) or goat anti-mouse CD206 (sc-34577, Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies at a concentration of 10 mg$mL -1 followed by AlexaFluor 488-conjugated anti-rat or anti-goat IgG secondary antibodies (Thermo Fisher Scientific). Immunofluorescence signals were visualized under a fluorescence microscope (Leica) and quantified using the ImageJ 2.0 imaging suite (U.S. National Institutes of Health, Bethesda, MD, USA). All signal counting procedures were performed by a blinded observer. Mean lipid droplet surface in adipose tissue H&E-stained samples was, in turn, quantified with ImageJ 2.0 (NIH).
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