The tissue sections were transferred directly into antigen retrieval solution (sodium citrate, pH 6.0) and then blocked with a mixture of 3% goat serum, 1% bovine serum albumin (BSA), and 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 1 h at room temperature. Subsequently, the sections (20 μm) were probed overnight with the primary antibodies at 4 °C and then incubated with the secondary antibodies for 1 h at room temperature. The cell nuclei were counterstained with DAPI and the images were recorded using confocal laser scanning microscope (FV3000, Olympus, Tokyo, Japan). The following primary antibodies were used: Endomucin (sc-65495, SANTA CRUZ, Dallas, TX, USA) and CD31 (ab28364, Abcam, Cambridge, UK). The secondary antibodies used included anti-rat Alexa Fluor 488 (4416, Cell Signaling, Boston, MA, USA) and anti-rabbit Alexa Fluor 649 (BS10034, Bioworld, Dallas, TX, USA).
Endomucin sc 65495
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Endomucin (sc-65495) is a lab equipment product offered by Santa Cruz Biotechnology. It is an antibody used in research applications. The core function of this product is to detect and identify the Endomucin protein in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (2 mentions)
Lsm 510 meta confocal microscope, by Zeiss (2 mentions)
Cd31 ab28364, by Abcam (1 mentions)
Fv3000, by Olympus (1 mentions)
Biotinylated antibodies, by Vector Laboratories (1 mentions)
Immu mount mounting medium, by Thermo Fisher Scientific (1 mentions)
Ng2 ab5320, by Merck Group (1 mentions)
Rapamycin, by Merck Group (1 mentions)
Alexa fluor 647, by Jackson ImmunoResearch (1 mentions)
Alexa fluor labeled secondary antibody, by Thermo Fisher Scientific (1 mentions)
αsma c6198, by Merck Group (1 mentions)
Cd45 30 f11, by BD (1 mentions)
Ab22552, by Abcam (1 mentions)
Ab19027, by Abcam (1 mentions)
Alexa fluor 488 conjugated anti gfp a21311, by Thermo Fisher Scientific (1 mentions)
Osterix, by Abcam (1 mentions)
Cathepsin k, by Abcam (1 mentions)
5 protocols using endomucin sc 65495
1
Immunohistochemistry of Endothelial Markers
2
Immunofluorescence Staining of Frozen Sections
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Unless otherwise stated, frozen sections were fixed in ice-cold acetone for 10 min followed by permeabilisation with 0.5% NP-40 for 10 min. Sections were blocked for 45 min with 1.0% bovine serum albumin (BSA) and 0.1% Tween 20 in PBS. Primary antibodies were incubated overnight at 4°C followed by incubation with a fluorescently conjugated secondary antibody for 1 h at room temperature (1:1000; Invitrogen). Primary antibodies against the following were used: α6-integrin (GoH3, Chemicon), NG2 (AB5320, Millipore), endomucin (sc-65495, Santa Cruz Biotechnology) (all 1:100).
3
Immunohistochemical Analysis of Vascular Markers
4
Tumor Vasculature Characterization Protocol
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Paraffin sections: Tumors were removed, fixed with PFA 4% for 24 h, left in PFA 1%, embedded into paraffin and sections (5 μm) were prepared.
Cryosections: Tumors were removed and frozen into TissueTek. 10 μm sections were prepared and fixed with formalin 4% (10 min, RT).
Stainings: Sections were blocked (1% BSA/PBS), incubated with primary antibodies (CD31, 553370, BD Pharmingen; endomucin, sc-65495 Santa Cruz; α-SMA C6198, Sigma) for 2 h, at RT or o/n at 4°C, washed, incubated with AlexaFluor-labeled secondary antibodies (45 min, RT, Life Technologies) and Hoechst 33342 (5 μg/ml) and mounted (Fluorsave Reagent, Calbiochem). Pictures were taken with a Zeiss LSM 510 META confocal microscope.
For evaluations of stainings, ImageJ and the particle counter plugin were used. Vessel number was determined by counting the number of vessels per mm2. Vessel size was determined by evaluating the area covered by vessels divided by the number of vessels per μm2. For the quantification of smooth muscle cell coverage of vessels, tumor sections were stained for CD31 and α-smooth muscle cell actin (α-SMA). Vessels with and without αSMA-staining were counted.
Cryosections: Tumors were removed and frozen into TissueTek. 10 μm sections were prepared and fixed with formalin 4% (10 min, RT).
Stainings: Sections were blocked (1% BSA/PBS), incubated with primary antibodies (CD31, 553370, BD Pharmingen; endomucin, sc-65495 Santa Cruz; α-SMA C6198, Sigma) for 2 h, at RT or o/n at 4°C, washed, incubated with AlexaFluor-labeled secondary antibodies (45 min, RT, Life Technologies) and Hoechst 33342 (5 μg/ml) and mounted (Fluorsave Reagent, Calbiochem). Pictures were taken with a Zeiss LSM 510 META confocal microscope.
For evaluations of stainings, ImageJ and the particle counter plugin were used. Vessel number was determined by counting the number of vessels per mm2. Vessel size was determined by evaluating the area covered by vessels divided by the number of vessels per μm2. For the quantification of smooth muscle cell coverage of vessels, tumor sections were stained for CD31 and α-smooth muscle cell actin (α-SMA). Vessels with and without αSMA-staining were counted.
5
Immunohistochemistry and In Situ Hybridization
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Immunohistochemistry of whole-mount samples or tissue sections was performed as described previously (Kubota et al., 2011 (link)). The primary monoclonal antibodies used were hamster anti-CD31 (MAB1398Z; 1:1,000; Chemicon), Nestin (Rat401; 1:200; BD Pharmingen), VEGFR2 (Avas12a1; 1:200; BD Pharmingen), and CD45 (30-F11; 1:500; BD Pharmingen). The primary polyclonal antibodies used were Alexa Fluor 488–conjugated anti-GFP (A21311; 1:500; Molecular Probes), Endomucin (sc-65495; 1:500; Santa Cruz), Osterix (ab22552; 1:500; Abcam), Cathepsin K (ab19027; 1:500; Abcam), and cleaved caspase 3 (9694; 1:200; Cell Signaling). Secondary antibodies used were Alexa Fluor 488–conjugated IgGs (A11034, A11006, A11055; 1:500; Molecular Probes) or Cy3/Cy5 DyLight549/DyeLight649-conjugated IgGs (711-165-152, 112-165-167, 127-165-160, 711-605-152, 112-605-167, 127-605-160; 1:500; Jackson ImmunoResearch). For nuclear staining, specimens were treated with DAPI (D-1306; Molecular Probes). For in situ hybridization, mandibular sections generated in RNAse-free conditions were hybridized with digoxigenin-labeled antisense RNA probes, as described previously (Kubota et al., 2011 (link)).
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