For immunofluorescence microscopy, frozen tissue sections (12-µm thick) were incubated with antibodies against the following primary antibodies: NG2 (1:100, Millipore, Temecula, CA, USA), PECAM-1 (1:100, Millipore), nitrotyrosine (1:100, Sigma-Aldrich), or βIII tubulin (1:100, Abcam, Cambridge, MA, USA) at 4℃ overnight. After several washes with PBS, the samples were incubated with tetramethyl rhodamine isothiocyanate- or fluorescein isothiocyanate-conjugated secondary antibodies (1:100, Zymed Laboratories, South San Francisco, CA, USA) for 2 hours at room temperature. Samples were mounted in a solution containing 4,6-diamidino-2-phenylindole (DAPI, Vector Laboratories Inc., Burlingame, CA, USA) for nuclei staining, and fluorescent signals were visualized using a confocal microscope (K1-Fluo, Nanoscope Systems Inc., Daejeon, Korea). Quantitative analysis of histological examinations was performed using ImageJ software.
Pecam 1
Manufactured by Merck Group
Sourced in United States
PECAM-1 is a cell surface glycoprotein that belongs to the immunoglobulin superfamily. It is primarily expressed on the surface of endothelial cells and plays a role in cell-cell adhesion. PECAM-1 is involved in the regulation of vascular permeability and the transendothelial migration of leukocytes.
Automatically generated - may contain errors
Lab products found in correlation
βiii tubulin, by Abcam (3 mentions)
Tetramethyl rhodamine isothiocyanate or fluorescein isothiocyanate conjugated secondary antibodies, by Zymo Research (2 mentions)
Oct media, by Electron Microscopy Sciences (2 mentions)
4 6 diamidino 2 phenylindole dihydrochloride dapi, by Merck Group (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Vector Laboratories (1 mentions)
Nitrotyrosine, by Merck Group (1 mentions)
Claudin 5, by Thermo Fisher Scientific (1 mentions)
α sma, by Merck Group (1 mentions)
Hsp70, by Abcam (1 mentions)
Caseviewer version 2, by 3DHISTECH (1 mentions)
Tcs sp8, by Leica (1 mentions)
Alexa fluor 594 goat anti rat igg, by Thermo Fisher Scientific (1 mentions)
α smooth muscle actin, by Merck Group (1 mentions)
Fv1000, by Olympus (1 mentions)
Monoclonal antibody against β actin, by Cell Signaling Technology (1 mentions)
Phospho smad1 5, by Cell Signaling Technology (1 mentions)
Phospho smad2, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Gradient gel, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
12 protocols using pecam 1
1
Immunofluorescence Microscopy of Frozen Tissue Sections
2
Immunohistochemical Analysis of Penile Tissues
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Penis tissues, MPGs, DRGs, and MCECs were fixed in 4% paraformaldehyde at 4°C for 30 minutes. Frozen tissue sections (12 µm thick), ex vivo samples, and MCECs were washed and incubated at 4°C overnight with antibodies against Hsp70 (Abcam; 1:100), Cse (1:100), PECAM-1 (Millipore, Burlington, MA, USA; 1:50), NG2 (Millipore; 1:50), α-SMA (Sigma-Aldrich; 1:100), phospho-eNOS (Invitrogen; 1:50), neurofilament (Sigma-Aldrich; 1:50), βIII tubulin (Abcam; 1:100), neuronal nitric oxide synthase (nNOS; Santa Cruz Biotechnology; 1:50), phospho-hHistone3 (Millipore; 1:50), and/or claudin-5 (Invitrogen; 1:100), as described in the text. After several washes with PBS, the samples were incubated with species-appropriate tetramethyl rhodamine isothiocyanate- or fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Zymed Laboratories, South San Francisco, CA, USA; 1:100) for 2 hours at room temperature. Samples were mounted in a solution containing DAPI, included to stain nuclei, and fluorescence signals were visualized using a confocal microscope (K1-Fluo; Nanoscope Systems, Inc., Daejeon, Korea). Quantitative histologic examinations were performed using Image J software.
3
Immunofluorescence analysis of cochlear vasculature
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The cochlear lateral wall tissues were collected and fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. Tissues were permeated and blocked with 0.3% Triton X-100/10% normal goat serum/PBS for 1 h and then incubated with PECAM1 (Millipore, Burlington, MA, USA), MAB 13982, IgG) antibody at a concentration of 1:200 in blocking solution overnight at 4 °C. After rinsing six times in PBS for 10 min, Alexa Fluor 594 goat anti rat IgG (A11007, 1:200, Thermo Fisher Scientific, Waltham, MA USA) was used as a secondary antibody for PECAM. The specimens were mounted on glass slides using 50% glycerol and observed using a confocal microscopy TCS SP8 (Leica Microsystems, Wetzlar, Germany). The thickness of the stria vascularis was measured, as described previously [104 (link)], using image analysis software (Case Viewer, version 2.1, 3DHISTECH, Ltd. Budapest, Hungary).
4
Immunohistochemical Analysis of Penile Tissue
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The penis tissue (N = 6 per group) and cultured MPG (N = 4 per group) were fixed in 4% paraformaldehyde for 24 hours at 4 °C as described previously42 (link). Frozen tissue sections (12-μm or 60-μm thick) were incubated with antibodies to smooth muscle α-actin (Sigma-Aldrich; 1:50), NG2 (Millipore, Temecula, CA, USA; 1:50), PECAM-1 (Millipore; 1:50), or βIII tubulin (Abcam, Cambridge, UK; 1:50) at 4 °C overnight. After several washes with PBS, the tissues were incubated with tetramethyl rhodamine isothiocyanate- or fluorescein isothiocyanate-conjugated secondary antibodies (Zymed Laboratories, South San Francisco, CA, USA) for 2 hours at room temperature. Samples were mounted. Signals were visualized and digital images were obtained with a confocal microscope (FV1000, Olympus, Tokyo, Japan). Quantitative analysis of histologic examinations was done with an image analyzer system (National Institutes of Health [NIH] Image J 1.34, http://rsbweb.nih.gov/ij/ ) and we analyzed the histologic data in a blinded manner.
5
Western Blot Analysis of Lung Proteins
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Lung tissues and cells were homogenized in lysis buffer with protease inhibitor cocktail (Roche, IN, USA)25 (link). After determining the protein concentration by DC protein assay kits (Bio-Rad, Hercules, CA, USA), total soluble proteins (20 μg) were separated on 4–12% gradient gels (Invitrogen, CA, USA)25 (link). The proteins were transferred to a nitrocellulose membrane (Amersham, Little Chalfont, UK). Nonspecific binding was blocked with 5% skim milk (BD, CA, USA) at room temperature for 1 h25 (link). Then, the protein blots were probed with specific primary antibodies (1:1000 dilutions) against phospho-Smad1/5 (Cell Signaling, Frankfurt, Germany), phospho-Smad2 (Millipore, MA, USA), phospho-Smad3 (Millipore, MA, USA), PECAM-1 (Millipore, MA, USA), and L- and S-endoglin (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. After incubating with host-specific secondary antibodies (goat anti-rabbit IgG-HRP) for 1 h at room temperature, the immunoreaction was detected with a chemiluminescent substrate kit (Thermo Scientific, Rockford, IL, USA) and quantitated by densitometric scanning of the X-ray film with SLB MyImager (UVP Inc, Upland, CA, USA)25 (link). The blots were normalized for protein loading by washing in stripping solution and reprobing with a monoclonal antibody against β-actin (Cell Signaling, Frankfurt, Germany)25 (link).
6
Immunohistochemical Analysis of Blood Vessels
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All samples were stored overnight in 10% formaldehyde solution and embedded in paraffin. Four microgram section was cut for immunohistochemistry and treated for antigen activation. Nonspecific binding sites were pre-blocked using 3% hydrogen peroxide for 30 minutes. The primary antibody used for detection of blood vessel was rabbit anti-human platelet endothelial cell adhesion molecule-1 (PECAM-1, 1:250, Millipore, Billerica, MA, USA). The primary antibody was incubated overnight at 4℃ and a secondary biotinylated anti-rabbit IgG (1:100, Promega, Sunnyvale, CA, USA) antibody was incubated following the primary incubation. Finally, the staining results were visualized using a standard DAB kit (Vector lab., Burlingame, CA, USA) according to the manufacturer's recommendation. Morphometric measurements and analysis were done with a semi-automatic dedicated software (ImageJ, http://rsb.info.nih.gov/ij ). The result of the morphometry was expressed as the PECAM-1 positive % area of the whole image.
7
Quantitative Vascular Structure Analysis
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The samples were stained and analysed according to established protocols (Kniebs et al., 2019 (link); Kreimendahl et al., 2019 (link)). The cells were stained with the fluorescent Vybrant™ dyes DiO, Dil and DiD (ThermoFisher Scientific) according to manufacturer´s instructions. Additionally, the samples to determine the influence of tumor-specific therapeutics were stained with mouse anti-CD31 (PECAM-1, 1:100; Sigma-Aldrich) and Alexa Fluor® 594 goat anti-mouse IgG (1:400, Acris Antibodies). The quantitative evaluation was performed with a two-photon laser scanning microscope, images processed and evaluated with Imaris 9.6.0 software (Bitplane Inc. South Winsor). The vascular structures were quantified according to their average volume, surface area, length, total number of vascular structures and branching points.
8
Cerebral Vascular Density Quantification
9
Immunocytochemical Analysis of Sox2 and CD31
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For immunocytochemistry, samples were fixed with 4% paraformaldehyde in PBS for 30 min at 37°C. Samples were permeabilized with 0.25% Triton X-100 in PBS (PBST) for 1 h at room temperature. The samples were then blocked with 5% bovine serum albumin (BSA) and 5% goat serum in PBST for 3 h at room temperature. Primary antibodies against sex determining region Y-box 2 (Sox2) (1:400, rabbit, Cell Signaling Technology, 23064S) and/or cluster of differentiation 31 (CD31) (1:200, mouse, PECAM-1, Sigma, P8590) were diluted in PBST containing 2.5% goat serum, added to samples, and incubated overnight at 4°C. The samples were then washed thrice with PBST, incubated with secondary antibodies [AlexaFluor488 (goat anti-rabbit, 1:500); AlexaFluor647 (goat anti-mouse, 1:500); and/or tetramethylrhodamine (TRITC) phalloidin 532 (F-actin, 1:500)], and counterstained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI) overnight at 4°C. Samples were washed thrice with PBST and imaged using a Leica SPE confocal microscope. Three independent biological replicates were used for each time point and variable (N = 3).
10
Cerebral Vascular Density Quantification
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WT (2 mo, n = 4; 10mo, n = 5) and mdx (2 mo, n = 4; 10 mo, n = 5) mice were deeply anesthetized with isoflurane after imaging. The brains were excised and fixed in 4% paraformaldehyde for 24 h at 4 °C. The tissue was transferred to 5% sucrose in PBS for 1 h followed by 20% sucrose overnight at 4 °C. Subsequently, the brains were rapidly frozen in OCT media (Electron Microscopy Sciences, Hatfield, PA) with liquid nitrogen. Tissues were sectioned at 10 µm slice thickness at the location corresponding to the MR imaging slice. A total of 7 sections were obtained from each brain. Indirect immunofluorescence against CD31 (PECAM-1, 1:100; Sigma Aldrich, St. Louis, MO) was performed to assess cerebral vascular density. DAPI (4′,6-diamidino-2-phenylindole dihydrochloride; Sigma Aldrich, St. Louis, MO) staining was used for nuclear identification. Stained tissue sections were photographed for the DAPI (blue) and PECAM-1 (red) channels. The number of PECAM-1-positive cells per field was quantified. All images were photographed at the same exposure times and magnification. The obtained values from all animals were averaged, and the results were expressed as the mean number of PECAM-1-positive cells per field ± SD.
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