Prostatic epithelial subpopulations were quantified as described (17 (link)). Briefly, individual prostates were dissociated and stained with the following antibodies purchased from eBioscience: CD31 (PECAM-1) (reference: 11-0311-85; conjugation: FITC; dilution 1:250), CD45 (reference: 11-0451-85; conjugation: FITC; dilution 1:250), TER-119 (reference: 11-5921-85; conjugation: FITC; dilution 1:250), CD49f (integrin α6) (reference: 12-0495-83; conjugation: PE; dilution 1:25), and Ly-6A/E (Sca-1) (reference: 17-5981-82; conjugation: APC; dilution 1:75), with DAPI. After selecting single, living cells, endothelial cells (CD31+), leukocytes (CD45+), and erythrocytes (TER-119+) were excluded, and epithelial subsets [luminal A/B (CD49f + Sca-1−); luminal-C (CD49fmedSca-1−); basal (CD49f+Sca-1−)] and stromal fibroblasts (CD49f−Sca-1+) were quantified. Data of individual mice were acquired using a BD LSR II flow cytometer and analyzed using the FlowJo software. To sort the epithelial subpopulations and stromal fibroblasts for subsequent protein analysis, dissociated prostates of three mice were pooled and sorted using a BD FACSAria Fusion flow cytometer.
Cd31 pecam 1
Manufactured by Thermo Fisher Scientific
Sourced in United States
CD31 (PECAM-1) is a cell surface glycoprotein that is primarily expressed on endothelial cells, platelets, and certain leukocyte subsets. It plays a role in cell-cell adhesion, and is involved in the regulation of angiogenesis and vascular permeability.
Automatically generated - may contain errors
Lab products found in correlation
Facsaria fusion flow cytometer, by BD (1 mentions)
Ter119, by Thermo Fisher Scientific (1 mentions)
Facsdiva software, by BD (1 mentions)
Facsaria sorter, by BD (1 mentions)
Cd140a pdgfra, by Thermo Fisher Scientific (1 mentions)
Paraformaldehyde (pfa), by Nacalai Tesque (1 mentions)
Ve cadherin, by Santa Cruz Biotechnology (1 mentions)
Biorevo bz 9000 fluorescence microscope, by Keyence (1 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iscript cdna synthesis kit, by Bio-Rad (1 mentions)
Purelink rna micro kit, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrometer, by Thermo Fisher Scientific (1 mentions)
Pparg, by Thermo Fisher Scientific (1 mentions)
Fabp4, by Thermo Fisher Scientific (1 mentions)
Taqman fast advanced mix, by Thermo Fisher Scientific (1 mentions)
Cryostat, by Leica (1 mentions)
Laminin, by Thermo Fisher Scientific (1 mentions)
Myhcslow ba f8, by Developmental Studies Hybridoma Bank (1 mentions)
Myhc2a sc 71, by Developmental Studies Hybridoma Bank (1 mentions)
Laminin sigma l9393, by Merck Group (1 mentions)
6 protocols using cd31 pecam 1
1
Quantification of Prostatic Epithelial Subpopulations
2
Isolation of Epidermal Progenitors from Mouse Skin
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Methods for preparing embryonic mouse back and head skin for fluorescence activated cell sorting (FACS) and purification of α6-high epidermal and hair bud progenitors have been previously described (Williams et al., 2011 (link)). Briefly, the skin of E14.5 and E17.5 embryos was dissected and either (E14.5) placed directly into a trypsin-EDTA solution at 37°C for 5 min on an orbital shaker, or (E17.5) first treated with the enzyme dispase (Gibco, 1:1 in PBS) overnight at 4°C prior to making the single cell suspension. Sorting buffer (PBS 5% FBS) was added to the suspension to neutralize trypsin. Single-cell suspensions were obtained by filtering through a 70 µM strainer and collected by centrifugation at 300 g for 5 min. Cell suspensions were washed three times and incubated with the appropriate antibodies for 30 min on ice. For FACS, we used the following antibodies (along with epifluorescent markers): α6-integrin (eBiosciences) to select for basal progenitors, CD140a (PDGFRA) (eBiosciences) to select against mesenchymal cells, CD31 (PECAM1) (eBiosciences) to select against platelets. DAPI was used to exclude dead cells. Cell isolations were performed on FACS Aria sorters running FACS Diva software (BD Biosciences).
3
Phenotypic Characterization of Differentiated Cells
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Cells were stained with phycoerythrin-conjugated monoclonal antibodies against
cell-surface markers, CD34 (1:100; eBioscience, CA), CD31 (PECAM-1) (1:100;
eBioscience, CA), CD45 (1:100; eBioscience, CA), and VE-cadherin (1:100; Santa
Cruz Biotechnologies, TX), to characterize the differentiated cell phenotypes.
The differentiated cells were fixed with 4% paraformaldehyde (PFA) (Nacalai
Tesque, Japan) for 10 min, permeabilized with Triton X-100 (0.1%) in
phosphate-buffered saline (PBS) for 10 min, and then incubated with bovine serum
albumin (BSA) (5%) for 10 min, followed by overnight incubation with the primary
antibodies in 5% BSA in PBS at 4°C. The next day, the cells were washed with PBS
twice, and the nuclei were stained with 4′,6-diamidino-2-phenylindole. Then, the
staining samples were inspected with a fluorescent microscope.
cell-surface markers, CD34 (1:100; eBioscience, CA), CD31 (PECAM-1) (1:100;
eBioscience, CA), CD45 (1:100; eBioscience, CA), and VE-cadherin (1:100; Santa
Cruz Biotechnologies, TX), to characterize the differentiated cell phenotypes.
The differentiated cells were fixed with 4% paraformaldehyde (PFA) (Nacalai
Tesque, Japan) for 10 min, permeabilized with Triton X-100 (0.1%) in
phosphate-buffered saline (PBS) for 10 min, and then incubated with bovine serum
albumin (BSA) (5%) for 10 min, followed by overnight incubation with the primary
antibodies in 5% BSA in PBS at 4°C. The next day, the cells were washed with PBS
twice, and the nuclei were stained with 4′,6-diamidino-2-phenylindole. Then, the
staining samples were inspected with a fluorescent microscope.
4
Immunocytochemical Staining of Cardiomyocytes
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Cultures were fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.05% Triton-X 100 for 5 min. After blocking nonspecific binding sites with 10% skim milk in PBS for 1 hr, cultures were immunocytochemically stained using primary antibodies against sarcomeric myosin heavy chain (sMHC) (1:20 dilution; MF20, Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), Nkx2.5 (1:100 dilution; Santa Cruz Biotechnology, Dallas, TX, USA), or CD31 (PECAM-1) (1:200 dilution; eBioscience), followed by secondary fluorescent antibodies as described previously52 (link). Samples were counterstained with Hoechst 33342 and examined under an Olympus IX-71 microscope (Olympus Japan, Tokyo, Japan). Photomicrographs were captured using an Olympus DP70 digital camera or a Biorevo BZ-9000 fluorescence microscope (KEYENCE Co., Osaka, Japan); images were analyzed using the BZ-II application.
5
Gene Expression Analysis of Bioprinted Tissues
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Gene expression was analyzed using real-time quantitative polymerase chain reaction (RT-qPCR). Adipose drop tissues at days 0, 7, and 14 of differentiation, as well as bioprinted fiber samples at days (before bioprinting) and at days 6 (myoblast fibers), 7 (endothelial fibers), or 14 (adipocyte fibers) were washed in PBS and total RNA extraction was carried out using the PureLink RNA Micro Kit (Invitrogen, Waltham, USA), with the DNAse step, following the manufacturer’s instructions. Samples’ RNA content was quantified with the NanodropTM spectrometer (N1000, Thermo Fisher Scientific, Waltham, USA). For RT-qPCR, the RNA samples were first submitted to reverse transcription into cDNA using iSCRIPT cDNA synthesis kit (Bio-Rad, Hercules, USA), before being amplified using Taqman probes and reagents (Taqman Fast Advanced Mix, Taqman gene expression assays (FAM): MYH2 (Assay ID: Bt03223147_gH), FABP4 (Assay ID: Bt03213820_m1), CD31/PECAM1 (Assay ID: Bt03215106_m1), PPARG (Assay ID: Bt03217547_m1), and PPIA (Assay ID: Bt03224615_g1), Thermo Fisher Scientific, Waltham, USA). The cDNA synthesis and RT-qPCR reactions were conducted using the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific, Waltham, USA) and the gene expression was normalized by PPIA as the housekeeping gene.
6
Skeletal Muscle Fiber Typing and Vascular Identification
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All frozen skeletal muscles were placed in a Leica cryostat and transverse sections of 10 μm were performed. As previously described by Gill et al.15 (link), sections were blocked for 45 min in 10% goat serum diluted in PBS 1X solution and incubated overnight at 4 °C in primary antibodies for the following Myosin heavy chain (MyHC) isoforms: MyHCSlow (BA-F8, 1:100 dilution; Developmental Studies Hybridoma Bank, Iowa City, IA), MyHC2A (SC-71, 1:100 dilution; Developmental Studies Hybridoma Bank), and Laminin (Sigma L9393, 1:200 dilution; Sigma-Aldrich, St. Louis, MO). Secondary antibodies were then incubated at a 1:2000 dilution, using Alexa Fluor Goat anti-Mouse IgG2b 647 (for MyHCSlow), Goat anti-Mouse IgG1 555 (for MyHC2A) and Goat anti-Rabbit 594 (for Laminin)15 (link). CD 31 (PECAM-1, 1:100; Thermo Fisher Scientific, Inc., MA) antibody was incubated with Laminin antibody and Alexa Fluor Goat anti-Rat 488 and Goat anti-Rabbit 594 were applied at 1:2000 dilution.
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