Immunohistochemical analysis of the rat livers was performed as previously described with slight modifications [27] (link). In brief, the rats were killed, and their livers were trimmed into a strip, soaked in 10% formalin, embedded in paraffin, and then sectioned. The liver sections were dewaxed, hydrated, subjected to heat-induced antigen retrieval, blocked in blocking buffer, and then incubated overnight at 4°C with an anti-alpha-smooth muscle actin (α-SMA) antibody (1:100; Dako, Denmark, Europe). The sections were then washed and further incubated with Super Enhancer and a poly-horseradish-conjugated reagent. The color was developed by incubating the sections with the 3,3′-diaminobenzidine and substrate reaction mixtures (1:38) as well as with hematoxylin. After washing the sections with water, the specific staining was visualized using light microscopy.
α sma antibody
Manufactured by Agilent Technologies
Sourced in Denmark, United States
The α-SMA (alpha-smooth muscle actin) antibody is a laboratory tool used to detect and quantify the presence of alpha-smooth muscle actin, a cytoskeletal protein found in vascular smooth muscle cells and other cell types. This antibody can be used in various research applications such as immunohistochemistry, Western blotting, and flow cytometry to study the expression and distribution of α-SMA in different tissues and cell samples.
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Lab products found in correlation
Ix81 inverted research microscope, by Olympus (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Anti desmin antibody, by Agilent Technologies (1 mentions)
Cox 2 polyclonal antibody, by Cayman Chemical (1 mentions)
Anti vimentin antibody, by Agilent Technologies (1 mentions)
Envision dual link system hrp dab, by Agilent Technologies (1 mentions)
Eclipse ts100, by Nikon (1 mentions)
Axioplan 2 confocal microscope, by Zeiss (1 mentions)
Jem 1011 microscope, by JEOL (1 mentions)
5 protocols using α sma antibody
1
Immunohistochemical Analysis of Rat Livers
2
Immunofluorescent Staining of α-SMA
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Cells were cultured on lysine-treated slides in a 6-well chamber prior to immunofluorescent staining. The cells were fixed in 4% paraformaldehyde for 15 min at room temperature. After washing with PBS, cells were permeabilized with 0.1% Triton X-100 for 30 min and blocked with 10% BSA in phosphate-buffered saline (PBS) for 60 min in a humidified chamber. Cells were incubated with α-SMA antibody (1:200 dilutions, Dako) overnight at 4°C. After washing with PBS, cells were incubated with TRITC-conjugated secondary antibody. Cell nuclei were counterstained with DAPI (Beyotime Biotechnology). Phase contrast and fluorescent microscopy was performed using an Olympus IX81 inverted research microscope (Olympus; Tokyo, Japan).
3
Characterization of Primary FM and FISS-Derived Cells
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To identify the origin of primary FM cells obtained from muscular tissue and FISS‐derived cells, ICCs were incubated with vimentin, desmin and anti‐alpha‐smooth muscle actin (α‐SMA) antibodies. The cells were seeded onto a 96‐well plate (4.5 × 103 cells/well) and incubated at 37°C for 24 h to reach 80%–90% confluence. After removal of the culture medium, the cells in each well were fixed with 80% acetone at −20°C for 10 min, air‐dried at RT and stored at −20°C for subsequent use. One hundred microliters of anti‐vimentin antibody (1:800 dilution) (Dako), anti‐desmin antibody (1:200 dilution) (Dako) and α‐SMA antibody (1:800 dilution) (Dako) were added to each well and incubated for 1 h at RT. After washing with PBS, the EnVision® + Dual Link System‐HRP (DAB+) (Dako) was used following the manufacturer's protocol. The images were evaluated using an inverted microscope (Eclipse TS 100; Nikon) by two pathologists. To quantify the positive cells in the tumour, five high‐power fields were randomly selected and captured and these pictures were analysed using ImageJ software (NIH). The ratio was calculated by dividing the positive cell count by the total cell count. To detect the expression of COX‐2 in FISS and FM cells, ICC was conducted as previously described. The primary antibody was replaced with a COX‐2 polyclonal antibody (Cayman) diluted at 1:200 in PBS.
4
Fibroblast-Tumor Cell Coculture Immunofluorescence
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Direct and indirect cocultures of fibroblast-GFP+ and tumor pancreatic cells were seeded on glass coverslips. Cells were washed twice with warm PBS, fixed in 4% paraformaldehyde and processed for immunofluorescence staining. First, cells were incubated overnight with mouse anti-α-Smooth Muscle Actin (α-SMA) antibody (n1584, Dako, Carpinteria, CA, USA) at 4 °C. After thorough washing with PBS, cells were incubated for 1 h with Alexa Fluor® 594-conjugated goat antimouse antibody and DAPI. Cells were mounted with Fluorsave™ Reagent (Calbiochem, San Diego, CA, USA) and examined in a Zeiss Axioplan 2 confocal microscope (Zeiss Microscopy, Göttinger, Germany).
5
Immunofluorescence and Transmission Electron Microscopy of Fibroblast-Tumor Cell Co-Cultures
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Direct and indirect co-cultures of broblasts-GFP + and tumor pancreatic cells were seeded on glass coverslips. Cells were washed twice with warm PBS, xed in 4% paraformaldehyde and processed for immuno uorescence staining. First, cells were incubated overnight with mouse anti-α-Smooth Muscle Actin (α-SMA) antibody (n1584, Dako, Carpinteria, CA) at 4ºC. After thorough washing with PBS, cells were incubated for 1h with Alexa Fluor® 594-conjugated goat anti-mouse antibody and DAPI. Cells were mounted with Fluorsave™ Reagent (Calbiochem, San Diego, CA) and examined in a Zeiss Axioplan 2 confocal microscope.
Transmission electron microscopy (TEM)
Cells were xed in 2.5% glutaraldehyde at pH 7.2 for 24h, and later in 1% OsO4 in a 0.1 M cacodylate buffer for 1h. Then, the samples were spinned to obtain pellets. The pellets were xed in 1% uranyl acetate for 30 minutes, were then dehydrated in a series of graded ethanol steps, and nally embedded in epoxy resin. Thin sections were performed and stained with toluidine blue. Ultrathin sections were obtained from representative areas and were double stained with lead citrate and uranyl acetate and viewed under a JEOL JEM-1011 microscope.
Transmission electron microscopy (TEM)
Cells were xed in 2.5% glutaraldehyde at pH 7.2 for 24h, and later in 1% OsO4 in a 0.1 M cacodylate buffer for 1h. Then, the samples were spinned to obtain pellets. The pellets were xed in 1% uranyl acetate for 30 minutes, were then dehydrated in a series of graded ethanol steps, and nally embedded in epoxy resin. Thin sections were performed and stained with toluidine blue. Ultrathin sections were obtained from representative areas and were double stained with lead citrate and uranyl acetate and viewed under a JEOL JEM-1011 microscope.
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