Total protein from whole lung or PLFs was isolated as previously described20 (link). Equal amounts of protein samples were separated on 4–20% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred to nitrocellulose membranes. The blots were blocked in blocking buffer (5% non-fat dry milk, 0.1% Tween 20 in TBS, pH 7.4) for 1 hour at room temperature, then incubated with rabbit anti-mouse α-SMA (Abcam, Cambridge, UK, 1:2,000), goat anti-mouse CXCR4 (Abcam 1:500), rabbit anti-mouse phosphor-Akt and pan Akt (Ser473) (Cell Signaling, Danvers, MA, 1:1,000), rabbit anti-mouse phospho-p44/42-MAPK (ERK1/2) and total p44/42-MAPK (ERK1/2) (Thr202/Tyr204) (Cell Signaling, Danvers, MA, 1:2,000), and rabbit anti-mouse GAPDH (Sigma, 1:50,000) at 4°C overnight in 5% bovine serum albumin in 0.1% Tween 20 in TBS. They were then washed and incubated for 1 hour at room temperature with an appropriate horseradish peroxidase–conjugated secondary antibody (Amersham Biosciences, Pittsburgh, PA, USA), washed, and visualized via enzyme-linked chemiluminescence using the SuperSignal West Pico kit (Pierce Biotechnology, Rockford, IL, USA).
Rabbit anti mouse α sma
Manufactured by Abcam
Sourced in United Kingdom
Rabbit anti-mouse α-SMA is a primary antibody that recognizes the alpha-smooth muscle actin (α-SMA) protein in mouse samples. α-SMA is a cytoskeletal protein found in vascular smooth muscle cells and is commonly used as a marker for this cell type.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Cytiva (2 mentions)
Rabbit anti mouse gapdh, by Merck Group (2 mentions)
Supersignal west pico kit, by Thermo Fisher Scientific (2 mentions)
Sirius red, by Abcam (2 mentions)
Dab staining, by Vector Laboratories (2 mentions)
Anti αsma ab, by Abcam (2 mentions)
Rabbit anti mouse desmin, by Thermo Fisher Scientific (2 mentions)
Rat anti mouse f4 80, by Thermo Fisher Scientific (2 mentions)
Rabbit anti mouse 4 hne, by Alpha Diagnostic (2 mentions)
Apotome microscope, by Zeiss (1 mentions)
Slowfade diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 488 or 568, by Thermo Fisher Scientific (1 mentions)
Axiozoom v16 microscope, by Hamamatsu Photonics (1 mentions)
Flash 4.0 v3, by Hamamatsu Photonics (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Rat anti mouse tgfβ1, by BD (1 mentions)
Species specific fluorochrome conjugated secondary antibodies, by Merck Group (1 mentions)
Rabbit anti mouse ki67, by Abcam (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)
Fluorescence microscope, by Olympus (1 mentions)
7 protocols using rabbit anti mouse α sma
1
Western Blot Analysis of Lung Proteins
2
Liver Fibrosis Immunohistochemistry Analysis
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Formalin-fixed frozen livers were stained with Sirius Red and anti-α-SMA Ab (Abcam). Immunohistochemistry was performed using DAB staining (Vector), and counterstaining with Hematoxilin. The following antibodies were used for immunostaining: rabbit anti-mouse α-SMA (Abcam, Cambridge, MA), rabbit anti-mouse Desmin (Thermo Fisher Scientific, Fremont, CA), rat anti-mouse F4/80 (eBioscience, San Diego, CA), or rabbit anti-mouse 4-HNE (Alpha Diagnostic Intl Inc., San Antonio, TX) antibodies, following incubation with Alexa Fluor ® secondary antibody. The images were taken using Nikon microscope, and analyzed by Image J.
3
Immunofluorescence Staining of Cornea
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Dissected corneas were fixed in 100% methanol at −20°C for 30 min and permeabilized with PBS containing 0.25% Triton X-100 (PBST). The cornea samples were blocked with 1% goat serum and 1% bovine serum albumin (BSA) in PBST for 30 min and stained with rabbit anti-mouse αSMA (Abcam, Cambridge, United Kingdom) overnight at 4°C. Following washing with PBST, corneas were stained with goat anti-rabbit 633 (Abcam) for 2 hours at room temperature and mounted flat in SlowFade Diamond Antifade Mountant (Thermo Fisher Scientific). Zeiss Apotome microscope was used for fluorescence imaging.
4
Immunocytochemistry Staining of MyoD and α-SMA
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For immunocytochemistry, formalin-fixed cells were rinsed with 0.05% Triton X-100 in PBS (PBS-1x) followed by incubation with 2% bovine serum albumin (BSA) in PBS-1x (PBS-1x/BSA) for blocking. Afterward, the cells were incubated with the desired primary antibody diluted in PBS-1x/BSA overnight at 4°C. Following two washes in PBS-1x, samples were incubated with secondary antibodies diluted in PBS-1x/BSA for 1 hour at room temperature. The following primary antibodies were used: rabbit anti-mouse MyoD (Invitrogen), rabbit anti-human MyoD (Invitrogen), and rabbit anti-mouse α-SMA (Abcam). 4′,6-Diamidino-2-phenylindole (DAPI) was used as a nuclear counterstain, and Alexa Fluor 488 or 568 served as the secondary antibody (all from Invitrogen). Imaging was performed by epifluorescence microscopy, consisting of a Xenon lamp, an Axio Zoom V16 microscope, and a Hamamatsu Flash 4.0 v3.
5
Western Blot Analysis of Protein Expression
6
Quantification of Cellular Markers in RSC96 and L929 Cells
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RSC96 cells were fixed with 4% paraformaldehyde, permeabilized with phosphate-buffered saline (PBS) containing 0.1% Triton X-100, blocked with PBS containing 0.1% Tween and 5% goat serum, and then stained with rabbit anti-rat p75 (1:100, Abcam), rabbit anti-rat Ki67 (1:100, Abcam), and rabbit anti-rat c-Jun (1:100, Abcam) primary antibodies. The coverslips were then sequentially labeled with species-specific fluorochrome-conjugated secondary antibodies and DAPI (Sigma-Aldrich, St. Louis, MO, United States). Samples were visualized using a fluorescence microscope (Olympus). The numbers of p75+ cells, Ki67+ cells, and c-Jun+ cells were counted at ×400 original magnification.
L929 cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, blocked with PBS containing 0.1% Tween and 5% goat serum, and then stained with rabbit anti-mouse α-SMA (1:100, Abcam) and rabbit anti-mouse Ki67 (1:100, Abcam) primary antibodies. The coverslips were then sequentially labeled with species-specific fluorochrome-conjugated secondary antibodies and DAPI (Sigma-Aldrich). Samples were visualized using a fluorescence microscope (Olympus). The numbers of α-SMA+ and Ki67+ cells were counted at ×400 original magnification.
L929 cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% Triton X-100, blocked with PBS containing 0.1% Tween and 5% goat serum, and then stained with rabbit anti-mouse α-SMA (1:100, Abcam) and rabbit anti-mouse Ki67 (1:100, Abcam) primary antibodies. The coverslips were then sequentially labeled with species-specific fluorochrome-conjugated secondary antibodies and DAPI (Sigma-Aldrich). Samples were visualized using a fluorescence microscope (Olympus). The numbers of α-SMA+ and Ki67+ cells were counted at ×400 original magnification.
7
Liver Fibrosis Immunohistochemistry Analysis
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