Colon tissues from mice were embedded in OCT compound (Sakura Finetek, Tokyo, Japan). Four-micrometer sections were fixed in 4% paraformaldehyde. After rinsing with PBS, sections were permeabilised and treated with blocking buffer (0.2% Triton X-100, 0.2% bovine serum albumin (BSA) and 0.1% normal goat serum in PBS). FITC-conjugated anti-IL-6 (eBioscience, Santa Clara, CA, USA), APC-conjugated anti-F4/80 (eBioscience) and Cy3-conjugated anti-α-SMA (Sigma-Aldrich) were used at 1 : 500 dilutions and incubated at 4 °C overnight, after which sections were washed with PBS and incubated with 4,6-diamidino-2-phenylindole (DAPI) for 5 min. After rinsing with PBS, the sections were mounted with mounting fluid and visualised under A1Rsi inverted Confocal Microscopy (Nikon).
Cy3 conjugated anti α sma
Manufactured by Merck Group
Sourced in United States
Cy3-conjugated anti-α-SMA is a laboratory reagent used for the detection and visualization of alpha-smooth muscle actin (α-SMA) in various biological samples. The product consists of a Cy3 fluorescent dye conjugated to an antibody specific to α-SMA, allowing for the identification and localization of this protein using techniques such as immunofluorescence microscopy.
Automatically generated - may contain errors
Lab products found in correlation
Mouse on mouse blocking kit, by Vector Laboratories (2 mentions)
Mouse anti α sma, by Thermo Fisher Scientific (2 mentions)
Rabbit anti p lrp1, by Thermo Fisher Scientific (2 mentions)
Qdot conjugated q705, by Thermo Fisher Scientific (2 mentions)
Lsm 710, by Zeiss (2 mentions)
Apc conjugated anti f4 80, by Thermo Fisher Scientific (1 mentions)
Oct compound, by Sakura Finetek (1 mentions)
Anti fcγ receptor, by BD (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Zen 2010, by Zeiss (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Dylight549 conjugated anti syrian hamster igg, by Immunotools (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Bovine serum albumin (bsa), by Fujifilm (1 mentions)
Pe conjugated anti cd31, by BioLegend (1 mentions)
Alexa fluor 647 conjugated anti goat igg, by Thermo Fisher Scientific (1 mentions)
Ab758, by Merck Group (1 mentions)
Guava easycyte, by Merck Group (1 mentions)
Blocking reagent, by Agilent Technologies (1 mentions)
C6198, by Merck Group (1 mentions)
7 protocols using cy3 conjugated anti α sma
1
Immunofluorescence Staining of Colon Tissues
2
Immunofluorescence Staining of Tumor Sections
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Tumors were harvested and fixed overnight at 4 °C in freshly prepared 4% paraformaldehyde (Merck Milipore) under agitation. Tumors were put overnight in 30% and 15% sucrose in 1xPBS, respectively and frozen in OCT. Serial sections were cut at a thickness of 8–12 μm using a cryotome. Cell layers of artLCMV-infected or uninfected human skin fibroblasts were fixed for 10 min with 4% PFA. Sections were blocked in PBS containing 10% FCS, 1 mg/ml anti-Fcγ receptor (BD Biosciences) and 0.1% Triton X-100 (Sigma). Sections were incubated with the following primary antibodies for 1–2 h at room temperature: anti-PDPN or anti-PDPN-bio (Clone eBio8.1.1, BioLegend, 1:200), anti-GFP (Aves, 1:1000), anti-mIL-33 (R&D systems, 1:500), AF647-conjugated anti-LCMV-NP (Clone BE0106, BioXcell, 1:200), Cy3-conjugated anti-αSMA (Clone 1A4, Sigma, 1:400), and FITC-conjugated CD90 (Clone 5E10, BioLegend, 1:100). Unconjugated antibodies were detected with the following secondary antibodies: AF488-conjugated anti-chicken-IgG (Thermofisher, 1:1000), Dylight549-conjugated anti-syrian hamster-IgG, AF647-conjugated anti-goat IgG, AF488-conjugated Streptavidin (all from Jackson Immunotools, 1:500 to 1:1000). Analysis was performed using a confocal microscope (LSM-710, Carl Zeiss) and the ZEN 2010 software (Carl Zeiss, v14.0.18.201). For image analysis Imaris version 9 (Bitplane) was used.
3
Flow Cytometric Characterization of Cell Markers
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Cells were detached using 0.2% EDTA/PBS. For VE-cadherin and CD31 staining, cells were blocked with 2% bovine serum albumins (BSA) (019–23293, Wako, Osaka, Japan)/PBS for 30 minutes. For αSMA and collagen type I staining, the cells were fixed in 4% paraformaldehyde (09154–85, Nacalai) for 10 minutes at room temperature, and blocked/permeabilized with 2% BSA/0.1% Triton X-100/PBS for 30 minutes. The cells were incubated with primary and subsequently secondary antibodies in 2% BSA/PBS for 1 hour at 4°C. Samples were analyzed with Guava easyCyte (Millipore, Billerica, MA, USA). Antibodies used were Cy3-conjugated anti-αSMA (1:500; C6198, Sigma), PE-conjugated anti-CD31 (1:50; 303105, Biolegend, San Diego, CA, USA), anti-collagen type I (1:80; AB758, Millipore) and Alexa Fluor 647-conjugated anti-goat IgG (1:200; A-21447, Thermo Fisher Scientific), and Alexa Fluor 647-conjugated anti-VE-cadherin (1:50; 561567, BD Biosciences, San Jose, CA, USA).
4
Quantitative Analysis of Microvascular Structure
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At POD 28, five mice from each group were euthanized to collect quadriceps/adductor muscles from ischemic hindlimbs. Harvested muscles were embedded in OCT compound (Tissue-Tek; Sakura Finetek Japan) and snap-frozen in liquid nitrogen. Frozen muscle sections (5-μm thickness) were air-dried and fixed with 4% paraformaldehyde (PFA) for 20 min at room temperature. After blocking with a blocking reagent (DAKO) including 1% Triton X-100, sections were incubated at 4 °C overnight with DyLite488-conjugated Lycopersicon esculentum Lectin (1:100; DL-1174, Vector Laboratories) for endothelial cell labeling and Cy3-conjugated anti-α-SMA (1:100; C6198, Sigma-Aldrich) for mural cell labeling. Microvessels were evaluated under a fluorescence microscope (BZ-X700; Keyence). Twenty different fields from four different cross sections were randomly chosen, and the number of microvessels per square millimeter was counted using cell counting software (BZ-II analyzer, Keyence); the average microvessel diameter was also obtained using the same microscopic field.
5
Renal Single Cell Analysis by Flow
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UUO (n = 6 mice) and sham (n = 6 mice) surgeries were performed, and renal single cells were isolated and analyzed by flow cytometry. Briefly, renal tissue was minced and placed into a cocktail of 0.25 mg/mL Liberase Blendzyme 3 (Roche Applied Science), 20 U/mL DNase I (Sigma-Aldrich) and shaken at 37 °C for 20 min. Subsequently, cells were passed through 40 μm nylon mesh and centrifuged (10 min, 200 g, 4 °C). Cells were stained and analyzed using flow cytometry. The following dyes and antibodies were used: APC-Cy7-conjugated anti-CD45, Alexa Fluor 700-conjugated anti-CD3 (both from BD Pharmingen), PerCP/Cy5.5-labeled anti-CD31, PE/Cy7-labeled anti-F4/80 (both from Biolegend), and Cy3-conjugated anti-α-SMA (Sigma). Stained single cells were resuspended in a staining buffer and immediately analyzed with a Becton Dickinson LSRII flow cytometer (BD Biosciences).
6
Immunohistochemical Analysis of Liver Fibrosis
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Paraffin-embedded livers (n=3–5 per time point) were sectioned into 4-μm thick slices. Standard immunohistochemistry was performed using anti-α-SMA (1:50), anti-Collagen I (1:100), and anti-fibrinogen (1:50) primary antibodies with an avidin–biotin complex-horseradish peroxidase developer. Double staining on sections used mouse anti-α-SMA (1:50) and rabbit anti-p-LRP1 (1:200) primary antibodies and Qdot-conjugated Q705 and Q605 secondary antibodies, respectively (Life Technologies). Triple staining was accomplished by adding goat anti-t-PA (1:150) labeled with biotinylated secondary and Streptavidin-Q655. Specificity of the α-SMA was confirmed using a mouse-on-mouse blocking kit (Vector Laboratories, Burlingame, CA, USA). Sequential double labeling of 4-μm thick OCT sections from LRPflox/flox; SM22-cre +/− or −/−, and t-PA+/+ or −/− mice (1:1 acetone:MeOH fix) used anti-LRP1 (1:50, American Diagnostica) with a Cy5-conjugated secondary antibody, followed by Cy3-conjugated anti-α-SMA (1:1000, Sigma-Aldrich).
7
Immunohistochemical Analysis of Liver Fibrosis
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