Jejunal tissue was analyzed by whole mount and cryostat section staining or GFP fluorescence using confocal microscopy as previously described [25 (link), 26 (link)]. Primary antibodies against the following antigens were used: anti-CACNA1G-C (rabbit, 1:50, SantaCruz, TX), anti-CACNA1G-N (rabbit, 1:50, Alomone Labs, Jerusalem, Israel and goat, 1:50, SantaCruz, TX), anti-PDGFR-alpha for mice (goat, 1:100, R&D system, MN), anti-PDGFR-alpha for humans (goat, 1:50, R&D system, MN), anti-PDGFR-beta (goat, 1:200, R&D system, MN), and anti-ACTA2 (rabbit, 1:200, Abcam, MA). Images were collected using the Fluoview FV10-ASW 3.1 Viewer software (Olympus, Tokyo, Japan) with an Olympus FV1000 confocal laser scanning microscope. Cryostat sections were also stained with β-galactosidase using LacZ Tissue Staining Kit (InvivoGen, San Diego, CA).
Anti acta2
Manufactured by Abcam
Sourced in Israel, United States
Anti-ACTA2 is a laboratory reagent that can be used to detect and quantify the ACTA2 (actin alpha 2, smooth muscle) protein. ACTA2 is a key component of the contractile apparatus in smooth muscle cells and is often used as a marker for these cell types. The antibody provided in this product can be used in various immunoassay applications to measure ACTA2 levels.
Automatically generated - may contain errors
Lab products found in correlation
Lacz tissue staining kit, by InvivoGen (1 mentions)
Anti cacna1g c, by Santa Cruz Biotechnology (1 mentions)
Fluoview fv10 asw 3.1 viewer software, by Olympus (1 mentions)
Anti pdgfr alpha, by R&D Systems (1 mentions)
Fv1000 confocal laser scanning microscope, by Olympus (1 mentions)
Anti ctnt, by Abcam (1 mentions)
Anti β catenin, by Proteintech (1 mentions)
Anti gata4, by Proteintech (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Sangon (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Bca assay, by Beyotime (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Loading buffer, by Thermo Fisher Scientific (1 mentions)
Anti col1a2, by Abcam (1 mentions)
Eyoecl plus kit, by Beyotime (1 mentions)
Bis tris acrylamide gel, by Thermo Fisher Scientific (1 mentions)
Lysis buffer, by Beyotime (1 mentions)
Anti bax, by Abcam (1 mentions)
Anti sm22, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
5 protocols using anti acta2
1
Immunohistochemical Analysis of Jejunal Tissue
2
Protein Expression and Nuclear Localization
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Total protein samples were prepared in radioimmunoprecipitation assay (RIPA) buffer; then, the protein concentration was determined by BCA assay (Beyotime). The total protein were separated by electrophoresis through Future PAGETM 4–12% (ACE, 11 Wells), then transferred the protein to PVDF membranes (Millipore, 0.45 µm), blocked with 8% skim milk and incubated with anti-PYGO2 (1:1000, Genetex), anti-NXK2.5 (1:1000; Invitrogen), anti-GATA4 (1:1000, Proteintech), anti-cTnT (1:1000, Abcam), anti-β-catenin (1:1000, Proteintech), anti-β-Tubulin (1:5000, ABcanol), anti-ACTA2 (1:1000, Abcam), anti-GAPDH (1:5000, ABcanol), and anti-β-ACTIN antibody (1:5000 dilution, ABcanol). The signal densities of the target protein bands were quantified and normalized to β-ACTIN or GAPDH using Image J. Nucleo-cytoplasmic isolation was performed according to the instructions of the Nuclear and Cytoplasmic Protein Extraction Kit (Sangon Biotech), followed by routine protein isolation and antibody incubation. As for the analysis of nuclear localization of β-catenin, we used the formula [(β-catenin/H3)-(Protein/GAPDH) + 1] (in both the vector and PYGO2 groups) to exclude for cytopasmic contamination.
3
Western Blot Analysis of Cell Signaling Proteins
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Cells were harvested after various treatments. Western blot analysis was conducted based on the published procedures [25 (link)]. Briefly, lysis buffer (Beyotime) was used to lyse cells. Lysates were mixed with loading buffer (Thermo Fisher), which was then boiled in boiling water for 8 min. The protein sample was loaded on 12% bis-tris-acrylamide gel (Thermo Fisher). Then, the protein bands were electrotransferred onto polyvinylidene fluoride (Millipore) and then immersed in 5% nonfat milk (Solarbio). Following that, the membranes were incubated with primary antibodies at 4°C overnight and secondary antibodies (peroxidase-conjugated IgG; 1:1,000; Abcam, Cambridge, UK) at 37°C for 2 h. Protein bands were visualized with eyoECL Plus Kit (Beyotime). GAPDH was employed as a control. Primary antibodies were anti-Ki67 antigen (anti-Ki67; 1:250; Abcam), anti-B-cell lymphoma-2 (anti-Bcl-2; 1:1,000; Abcam), anti-BCL2-associated x protein (anti-Bax; 1:1,000; Abcam), anti-Cleaved poly (ADP-ribose) polymerase (PARP) (anti-Cleaved PARP; 1:1,000; CST, Boston, MA, USA), anti-collagen typeI2 (anti-COL1A2; 1:1,000; Abcam), anti-actin alpha 2 (anti-ACTA2; 1:500; Abcam), and anti-GAPDH (1:1,000; CST).
4
Western Blot Antibody Validation
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The utilized antibodies were the following: anti‐ACTA2 (Abcam, Ca# ab32575) at the dilution of 1:2,000, Anti‐CNN1 (Santa Cruz, Ca# sc‐136987) at the dilution of 1:500, Anti‐SM22 (Santa Cruz, Ca# sc‐53932) at the dilution of 1:500, and anti‐GAPDH (Santa Cruz, Ca# sc‐32233) 1:2,000. The secondary antibodies were the following: Goat anti‐Rabbit IgG (H + L) Cross‐Adsorbed Secondary Antibody, HRP (Life Technologies, Ca# G‐21234), Goat anti‐Mouse IgG (H + L) Cross‐Adsorbed Secondary Antibody, HRP (Life Technologies, Ca# G‐21040), at the dilution of 1:5,000.
5
Immunohistochemical and Immunofluorescence Analysis
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The specimens were fixed with 4% paraformaldehyde (PFA) and embedded in paraffin after tissue processing. After deparaffinization, rehydration, and antigen retrieval, 2-µm-thick sections were blocked with 10% normal donkey serum for 1 h at room temperature and stained with primary antibodies. The following primary antibodies were used in immunohistochemical staining: anti-SDC4 (1:500; Proteintech, Wuhan, China) and anti-GPC3 (1:500; Proteintech, Wuhan, China). Biotinylated anti-rabbit (Beyond, Shanghai, China) antibody was used as secondary antibodies according to the manufacturer's protocol. 3,3N-Diaminobenzidine Tertrahydrochloride (Beyond, Shanghai, China) was used as a chromogen according to the manufacturer's protocol. For double-immunofluorescence analysis, sections were pretreated in the same way as for IHC. The following primary antibodies were used: anti-SDC4 (1:50; mouse; Santa Cruz Biotechnology) and anti-KRT19 (1:500; rabbit; Abcam); anti-GPC3 (1:500; rabbit; Abcam) and anti-ACTA2 (1:500; mouse; Abcam). All slides were incubated with primary antibody overnight, which was diluted with 10% normal donkey serum after 1-h serum blocking. After DAPI (4’6-diamidino-2-phenylindole) staining, images were acquired using an Olympus BX51 microscope.
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