One middle and one distal section from each xenograft were fixed in 10% NBF and embedded in paraffin, along with both proximal and distal sections of the transplant area belonging to the porcine native aorta. The specimens were sliced in 5 μm sections and stained with haematoxylin and eosin (HE), Masson's trichrome (MT, Sigma), Miller's elastic counterstained with Van Gieson (EVG, Atom Scientific), and toluidine blue according to the manufacturer's instructions (Sigma). For immunohistochemistry, the specimens were stained with anti-α smooth muscle actin antibody (α-SMA) (Abcam, UK), and anti-mast cell tryptase antibody (Abcam, UK). All samples were compared to control sections from either the infrarenal sheep aorta prior to decellularization or the infrarenal porcine aorta removed during the transplantation procedure.
Anti α smooth muscle actin α sma antibody
Manufactured by Abcam
Sourced in United States
Anti-α-smooth muscle actin (α-SMA) antibody is a laboratory reagent used for the detection and localization of α-smooth muscle actin in biological samples. It is a primary antibody that specifically binds to α-SMA, a key marker of smooth muscle cells.
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Lab products found in correlation
Anti vimentin antibody, by Abcam (2 mentions)
Toluidine blue, by Merck Group (1 mentions)
Anti mast cell tryptase antibody, by Abcam (1 mentions)
Masson s trichrome mt, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Anti fasn antibody, by Proteintech (1 mentions)
Anti c myc antibody, by Cell Signaling Technology (1 mentions)
Anti n cadherin antibody, by Abcam (1 mentions)
Anti rock1 antibody, by Abcam (1 mentions)
Anti rock2 antibody, by Abcam (1 mentions)
Anti rhoa antibody, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca kit, by Beyotime (1 mentions)
Anti mac3 antibody, by BD (1 mentions)
Anti neutrophil antibody, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Aspen (1 mentions)
Anti pcna antibody, by Abcam (1 mentions)
Alexa 488 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pas kit, by Merck Group (1 mentions)
9 protocols using anti α smooth muscle actin α sma antibody
1
Histological Analysis of Xenograft Aortic Tissue
2
Euthanasia and Tissue Analysis
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Euthanasia was performed on the 90th and 180th day after operation ( pentobarbital sodium overdose; 90 mg kg -1 ). The long tissue bands of 4 cm, including the material, materialhost tissue interface and adjacent host tissue, were removed from the abdominal wall of the implanted material. After 10% formalin fixation, 5 mm paraffin sections, haematoxylin-eosin staining and Masson staining were prepared and observed under a light microscope.
The muscle regeneration and inflammatory cell infiltration of different repair materials were analysed by immunohistochemical staining. Anti-α-smooth muscle actin antibody (α-SMA, 1 : 400, Abcam) and anti-CD163 antibody (1 : 100, ZSGB-Bio) were used to detect actin positive cells and inflammatory reaction. The samples were magnified and quantified by a double-blind method using IMAGE-Pro Plus software. Percentage of positive area = Positive area/Total tissue area.
The muscle regeneration and inflammatory cell infiltration of different repair materials were analysed by immunohistochemical staining. Anti-α-smooth muscle actin antibody (α-SMA, 1 : 400, Abcam) and anti-CD163 antibody (1 : 100, ZSGB-Bio) were used to detect actin positive cells and inflammatory reaction. The samples were magnified and quantified by a double-blind method using IMAGE-Pro Plus software. Percentage of positive area = Positive area/Total tissue area.
3
Prostate Cancer Metabolism Profiling
4
Western Blot Analysis of Epithelial-Mesenchymal Transition
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Total protein was extracted from SRA01/04 cells after lysis on ice using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using a BCA kit (Beyotime Institute of Biotechnology). A total of 30 µg protein samples per well were then transferred to PDVF membranes after resolving using 10% SDS-PAGE gels. Subsequently, the membranes were washed, blocked with 5% skimmed milk for 1 h and then incubated with the following primary antibodies at 4˚C overnight: Anti-E-cadherin antibody (1:10,000; cat. no. ab40772; Abcam), anti-N-cadherin antibody (1:5,000; cat. no. ab76011; Abcam), anti-Vimentin antibody (1:1,000; cat. no. ab92547; Abcam), anti-α-smooth muscle act in (α-SMA) antibody (1:1,000; cat. no. ab265588; Abcam), anti-RhoA antibody (1:5,000; cat. no. ab187027; Abcam), anti-ROCK1 antibody (1:1,000; cat. no. ab92547; Abcam) or anti-ROCK2 antibody (1:1,000; cat. no. ab134181; Abcam). After washing with PBS, the membranes were incubated with the HRP-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; cat. no. ab6721; Abcam) for 2 h at room temperature. All antibodies utilized in the present study were purchased from Abcam. GAPDH was used as the loading control. The protein blots were visualized using enhanced chemiluminescence (ECL) reagent and densitometry analysis of the bands was performed using ImageJ (National Institutes of Health).
5
Wound Healing Histological Analysis
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Mice were euthanized via the IP injection of a lethal dose of pentobarbital sodium on days 3, 7, 11, or 14 after wounding. The wound and the surrounding intact skin were harvested, and each sample of wound tissue and the surrounding intact skin was bisected at the center of the wound. The wound samples were stapled onto polypropylene sheets to prevent overcontraction, before being fixed in 4% paraformaldehyde for 18 hours. The samples were then dehydrated in a graded alcohol series, cleaned in xylene, and embedded in paraffin, before 5-μm serial paraffin sections were prepared. At least 6 serial sections from near to the center of the wound were obtained per wound and stained according to the following methods [17 (link)]. Five-μm thick sections were subjected to hematoxylin-eosin (HE) or azan staining or were immunohistologically stained with anti-neutrophil antibody to detect neutrophils (1 : 100; Abcam Japan, Tokyo, Japan), anti-Mac-3 antibody to detect macrophages (1 : 100; BD Pharmingen, Tokyo, Japan), or anti-α-smooth muscle actin (α-SMA) antibody to detect myofibroblasts (1 : 300; Abcam Japan, Tokyo, Japan). Negative control slides were obtained by omitting each primary antibody.
6
Histological Evaluation of Wound Healing
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The wound samples were harvested and embedded on day 15 after the rats euthanization. Then the wound sections were stained with H&E and masson staining kits to evaluate wound beds and collagen accumulation. The embedded wound sections were deparaffinized and incubated with the anti -PCNA antibody (Abcam, 1:200) overnight at 4 °C, and then incubated with an HRP-conjugated secondary antibody (Aspen, 1:400) for 1 hour at room temperature. The wound samples stored at −80 °C were initially embedded with OCT and sliced into a 4-μm-thick section, then incubated with an anti-α-smooth muscle actin (α-SMA) antibody (Abcam, 1:200) at 4 °C overnight, and subsequently incubated with an Alexa488-conjugated secondary antibody (Invitrogen, 1:400) for 1 hour at room temperature, and finally stained with the 4ʹ,6-diamidino2-phenylindole (DAPI, Sigma-Aldrich).
7
Immunohistochemical Analysis of PDGF-C in Mice Kidneys
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The kidneys of the mice were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Then, 4 μm sections were used for Periodic acid‐Schiff (PAS) staining and immunohistochemical analysis. Immunohistochemical analyses with polyclonal anti‐PDGF‐C antibody (R & D systems, Minneapolis, MN) were carried out as described (Makino et al. 2003 ). For semiquantitative analyses, 30 glomeruli were used to count PDGF‐C‐positive cells to determine the number of positive cells per glomerulus and the ratio of positive cells to the total number of cells. Immunofluorescent analyses were performed by incubation with polyclonal anti‐PDGF‐C antibody (Santa Cruz), anti–α‐smooth muscle actin (α‐SMA) antibody (Abcam), monoclonal anti‐CD34 antibody (Abcam), or polyclonal anti‐nephrin antibody (PROGEN Biotechnik GmbH, Heidelberg, Germany) followed by fluorescence‐conjugated secondary antibodies. Staining with 4,6‐diamidino‐2‐phenylindole (DAPI) was performed to identify the nucleus.
8
Immunofluorescence Staining of Corneal Markers
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EDTA, Glycerin, 4′,6-diamidino-2-phenylindole I (DAPI), bovine serum albumin (BSA), mouse anti-type IV collagen antibody, mouse anti-human collagen VII antibody, and PAS kit were from Sigma (St. Louis, MO, US). Dulbecco’s modified Eagle medium (DMEM) without phenol red was from Invitrogen (Eugene, OR, US). Mouse anti-laminin 5 antibody, anti-cytokeratin 12 (K12) antibody, anti-cytokeratin 19 (K19) antibody, anti-vimentin antibody, anti-α smooth muscle actin (α-SMA) antibody, and mouse anti-macrophage monoclonal antibody were from Abcam (Cambridge, MA, US). FITC-conjugated anti–mouse IgG was from DAKO (Denmark).
9
Histological Examination of Peritoneal Tissues
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Histology and immuno uorescence staining of 4-μm para n sections from the anterior or posterior peritoneal tissues were performed as described previously [21] . For identi cation of the HPMCs, the cells from the PDE were stained for CK 18 and vimentin. For con rmation of the expression of Nestin in the peritoneum, human peritoneum samples obtained from patients undergoing abdominal surgery were assessed by immuno uorescence staining. The following primary antibodies were used: rabbit monoclonal anti-vimentin antibody (Abcam, Cambridge, MA, USA), mouse monoclonal anti-CK 18 antibody (Abcam, Cambridge, MA, USA), rabbit polyclonal anti-collagen I (Col-I) antibody (Abcam, Cambridge, MA, USA), and anti-α-smooth muscle actin (α-SMA) antibody (Abcam, Cambridge, MA, USA).
Hematoxylin-eosin staining and Masson's staining.
The parietal peritoneum specimens from the 3 groups of mice were soaked in paraformaldehyde solution and then prepared for hematoxylin-eosin and Masson's staining as previously described [22] .
Hematoxylin-eosin staining and Masson's staining.
The parietal peritoneum specimens from the 3 groups of mice were soaked in paraformaldehyde solution and then prepared for hematoxylin-eosin and Masson's staining as previously described [22] .
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