All primary and secondary antibodies were dissolved in PBS (1:200) prior to labeling. The PBECs, astrocytes and pericytes were fixed in 4% paraformaldehyde and blocked in PBS supplemented with 0.2% Triton-X-100 and 3% bovine serum albumin for 1 hour. The PBECs were stained with polyclonal rabbit anti-claudin-5 (Sigma-Aldrich, cat. no. SAB4502981, lot 310145) and polyclonal rabbit anti-ZO-1 (Invitrogen, cat. no. 617300, lot 1087989A). Mixed glial cells were stained with rabbit anti-glial fibrillary acidic protein (GFAP)(DAKO, DK, cat. no. Z0334, lot 20003791) and Texas Red labelled Lycopersicon Esculentum (Tomato) Lectin (Vector Labs, Peterborough, United Kingdom, cat. no. TL1176, lot W0812). Pericytes were stained with monoclonal mouse anti-α-smooth muscle actin (α-SMA) (Sigma-Aldrich, cat. no. A5228, lot 091M4832), polyclonal rabbit anti-ZO-1 and rabbit anti-platelet-derived growth factor receptor-beta (PDGFR-β) (Santa Cruz, cat.no.Sc-432, lot K1113). For detection, the cells were subsequently stained with goat anti-rabbit Alexa 488 or goat anti-mouse Alexa 585 (Invitrogen) as the secondary antibodies. All cells were counterstained with DAPI. The Millicell membranes were cut out of the inserts and mounted on glass slides in fluorescent mounting media (Dako, Denmark) and cover slips were placed upon the membranes.
Monoclonal mouse anti α smooth muscle actin α sma
Manufactured by Merck Group
Monoclonal mouse anti-α-smooth muscle actin (α-SMA) is a lab equipment product used in research and diagnostics. It is a primary antibody that recognizes the α-smooth muscle actin protein, which is a marker for smooth muscle cells. This product can be used in various immunodetection techniques, such as immunohistochemistry, immunocytochemistry, and Western blotting, to identify and study smooth muscle-containing tissues and cells.
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Lab products found in correlation
Pdgfr β, by Santa Cruz Biotechnology (1 mentions)
Fluorescent mounting media, by Agilent Technologies (1 mentions)
Polyclonal rabbit anti zo 1, by Santa Cruz Biotechnology (1 mentions)
Polyclonal rabbit anti zo 1, by Thermo Fisher Scientific (1 mentions)
Glial fibrillary acidic protein (gfap), by Agilent Technologies (1 mentions)
Fibronectin, by Merck Group (1 mentions)
2 protocols using monoclonal mouse anti α smooth muscle actin α sma
1
Multicolor Immunostaining of Brain Cells
2
Immunocytochemical Analysis of Smooth Muscle Cells
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The purity of cells was assessed by immunocytochemical staining with a specific smooth muscle cell marker, monoclonal mouse anti-αsmooth muscle actin (α-SMA; Sigma-Aldrich). All cells were strongly positive for α-SMA (data not shown).
Myometrial and leiomyoma cells were seeded in chamber tissue culture slides and allowed to divide. Cells were washed three times with PBS, treated with 0.2% Triton X-100 in PBS for 5 min, and washed three times with PBS. To inhibit endogenous peroxidase activity, cells were | 5 incubated for 10 min with 3% hydrogen peroxide in deionized water.
Cells were washed three times with PBS, and to block nonspecific background, cells were incubated for 20 min at RT with normal horse serum, diluted 1:75 in 1% bovine serum albumin in PBS. Cells were then incubated with fibronectin (Sigma-Aldrich) monoclonal mouse antibody at 1:600 for 1 hr at RT. After washing with PBS, cells were incubated with biotinylated anti-mouse immunoglobulin G made in horse diluted 1:200. The peroxidase Avidin-Biotin Complex method was performed for 1 hr at RT using 3,3′-diaminobenzidine as chromogen. Sections were counterstained in Mayer's hematoxylin, dehydrated, and mounted with Eukitt solution (Bio-Optica, Milan, Italy).
Myometrial and leiomyoma cells were seeded in chamber tissue culture slides and allowed to divide. Cells were washed three times with PBS, treated with 0.2% Triton X-100 in PBS for 5 min, and washed three times with PBS. To inhibit endogenous peroxidase activity, cells were | 5 incubated for 10 min with 3% hydrogen peroxide in deionized water.
Cells were washed three times with PBS, and to block nonspecific background, cells were incubated for 20 min at RT with normal horse serum, diluted 1:75 in 1% bovine serum albumin in PBS. Cells were then incubated with fibronectin (Sigma-Aldrich) monoclonal mouse antibody at 1:600 for 1 hr at RT. After washing with PBS, cells were incubated with biotinylated anti-mouse immunoglobulin G made in horse diluted 1:200. The peroxidase Avidin-Biotin Complex method was performed for 1 hr at RT using 3,3′-diaminobenzidine as chromogen. Sections were counterstained in Mayer's hematoxylin, dehydrated, and mounted with Eukitt solution (Bio-Optica, Milan, Italy).
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