For the preparation of frozen sections from non-fixed and undecalcified hard tissues, Kawamoto’s film methods were used. Samples were freeze-embedded with super cryoembedding medium (SECTION-LAB Co. Ltd., Hiroshima, Japan) and cut in thickness of 5 μm after mounting the adhesive film onto the sample surface. Samples were then immediately fixed with 4% paraformaldehyde (PFA) for 20 min and stained with hematoxylin and eosin. For immunohistological analysis, the specimens were incubated with the anti-CD146 antibody (Abcam, San Francisco, CA, USA), or the isotype IgG (Abcam) at 4 °C overnight after blocking with 5% goat serum (Life Technologies, Gaithersburg, MD, USA). After washing, the specimens were incubated with secondary antibody Alexa Fluor 488 donkey anti-rabbit IgG (Life Technologies) for 60 min at room temperature. All images were taken by fluorescence microscope (Biozero BZ-X700, Keyence, Osaka, Japan).
Anti cd146 antibody
Manufactured by Abcam
Sourced in United States
Anti-CD146 antibody is a laboratory reagent used in research applications. It binds to the CD146 protein, also known as MUC18 or MCAM, which is expressed on the surface of various cell types. The antibody can be used to detect and study the expression of CD146 in different biological samples.
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Lab products found in correlation
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (2 mentions)
Goat serum, by Thermo Fisher Scientific (1 mentions)
Isotype igg, by Abcam (1 mentions)
Alexa fluor 488 donkey anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Biozero bz x700, by Keyence (1 mentions)
Alexa fluor conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Anti brdu antibody, by Novus Biologicals (1 mentions)
Rabbit anti cd34, by Abcam (1 mentions)
Envision kit, by Agilent Technologies (1 mentions)
Anti pax7antibody, by Abcam (1 mentions)
Anti mhc antibody, by Abcam (1 mentions)
Triton x 100, by Thermo Fisher Scientific (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Anti ssea4, by Merck Group (1 mentions)
Anti cd146, by Abcam (1 mentions)
Biozero bz 8000, by Keyence (1 mentions)
6 protocols using anti cd146 antibody
1
Frozen Section Preparation of Undecalcified Hard Tissues
2
Evaluating MSC Stemness in Hydrogels
3
BrdU and CD146 Expression in Wound Healing
4
Immunohistochemical Evaluation of TMEM164 in Human Liver Tissue
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Immunohistochemistry was performed using an Envision Kit (Dako, Carpinteria, CA). Human specimen slides were subjected to immunohistochemistry using mouse anti-alpha smooth muscle actin (Dako) and rabbit anti-CD34 (Abcam, Cambridge, MA) monoclonal primary antibodies and hematoxylin and eosin staining.
TMEM164 expression in human liver tissue was evaluated using a rabbit anti-TMEM164 polyclonal antibody (Abcam). Specimens with vascular endothelial cells that were stained more strongly than other constituent cells in both tumor and nontumor tissues were determined to have high staining intensity.
Double fluorescent staining was performed using a rabbit anti-TMEM164 polyclonal antibody and Alexa 488 FITC-conjugated rabbit anti-IgG, and mouse polyclonal anti-CD31 (Abcam) or anti-CD146 antibody (Abcam) and Alexa 488 FITC-conjugated rabbit anti-IgG as primary and secondary antibodies. Each of these antibodies was mixed and treated at 37°C for 24 hours. Culture slides were mounted and stained using DAPI. All images were obtained using a BIOREVO BZ-X810 fluorescence microscope system (Keyence, Osaka, Japan).
TMEM164 expression in human liver tissue was evaluated using a rabbit anti-TMEM164 polyclonal antibody (Abcam). Specimens with vascular endothelial cells that were stained more strongly than other constituent cells in both tumor and nontumor tissues were determined to have high staining intensity.
Double fluorescent staining was performed using a rabbit anti-TMEM164 polyclonal antibody and Alexa 488 FITC-conjugated rabbit anti-IgG, and mouse polyclonal anti-CD31 (Abcam) or anti-CD146 antibody (Abcam) and Alexa 488 FITC-conjugated rabbit anti-IgG as primary and secondary antibodies. Each of these antibodies was mixed and treated at 37°C for 24 hours. Culture slides were mounted and stained using DAPI. All images were obtained using a BIOREVO BZ-X810 fluorescence microscope system (Keyence, Osaka, Japan).
5
Immunocytochemical Quantification of MDSCs
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Immunocytochemistry cellular immunofluorescence was conducted as previously described [41 (link)]. The MDSCs were first fixed in 4% PFA (Sigma) and permeabilized with 1% Triton X-100 (Invitrogen). The cells were incubated with an optimal concentration of anti-MHC antibody, anti-CD146 antibody, anti-PAX7 antibody, and anti-PW1 antibody (1:200, all from Abcam) overnight at 4 °C, incubated with an Alexa Fluor 488 or 546 secondary antibody (1:800, Invitrogen), followed by rinsing three times with PBS. Then, cell nuclei were stained with Dapi (Invitrogen) prior to being imaged by fluorescent microscope (Olympus). The number, length, diameter, and maturation index data of myotubes were obtained from at least three images of each sample using ImageJ software, and cell positive rate of each marker was calculated by normalizing the positive cell number to the total cell number.
6
Immunostaining of Cryosections and DPCs
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Immunostaining of cryosections was performed by initial blocking with 10% normal goat serum, followed by incubation with anti-CD-146 antibody (Abcam, Cambridge, MA, USA) or IgG (Abcam), wash, and incubation with second antibody AlexaFluor 488 conjugated IgG (Invitrogen).
DPCs were seeded onto 96-well plates and cultured for 24 hours in basal medium. The cells were subsequently fixed in 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with PBS containing 0.25% Triton X-100 for 10 minutes, blocked with 10% normal goat serum, and incubated with primary antibodies anti-STRO-1 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-SSEA-4 (Millipore, Billerica, MA, USA), or anti-CD146 (Abcam) for 1 hour or overnight. In negative controls, primary antibodies were replaced by an appropriate isotype-matched negative IgM or IgG. Cells were incubated with AlexaFluor 488 conjugated anti-mouse IgM (Invitrogen) or IgG (Invitrogen).
Cell nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI) (Invitrogen). Analysis of immunofluorescence staining was performed under a microscope (Biozero BZ-8000; Keyence Corp., Osaka, Japan).
DPCs were seeded onto 96-well plates and cultured for 24 hours in basal medium. The cells were subsequently fixed in 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with PBS containing 0.25% Triton X-100 for 10 minutes, blocked with 10% normal goat serum, and incubated with primary antibodies anti-STRO-1 (Developmental Studies Hybridoma Bank, Iowa City, IA, USA), anti-SSEA-4 (Millipore, Billerica, MA, USA), or anti-CD146 (Abcam) for 1 hour or overnight. In negative controls, primary antibodies were replaced by an appropriate isotype-matched negative IgM or IgG. Cells were incubated with AlexaFluor 488 conjugated anti-mouse IgM (Invitrogen) or IgG (Invitrogen).
Cell nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI) (Invitrogen). Analysis of immunofluorescence staining was performed under a microscope (Biozero BZ-8000; Keyence Corp., Osaka, Japan).
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