Double-label immunofluorescent staining for lectin was performed to detect angiogenesis around the penumbra region on day 7 after occlusion. Rats were anesthetized at 7 days after the operation, and the brains were transcardially perfused with NS followed by 4% paraformaldehyde. After gradient elution with sucrose, the brains were quickly frozen and cut into 6 µm coronally thick sections. These sections were permeabilized with 0.5% Triton X-100 for 5 min and blocked with 10% donkey serum for 1 h. Then, sections were incubated with anti-BrdU (1 : 500, Abcam NBP2-29414) and anti-CD34 (1 : 100, Santa Cruz Biotechnology, USA) overnight at 4°C. Next, the sections were briefly washed with PBST and incubated with donkey anti-mouse secondary antibody (1 : 400, Abcam, United States) for 1 h at 37°C. After counterstaining with 4,6-diamidino-2-phenylindole (DAPI; Solarbio, Beijing, China), the double-stained sections on the right side were observed and photographed using a fluorescence microscope, and analysis was conducted with the ImageJ software (Rawak Software Inc., Germany).
Anti cd34
Manufactured by Santa Cruz Biotechnology
Sourced in United States, United Kingdom
Anti-CD34 is a laboratory reagent used for the detection and analysis of the CD34 cell surface antigen. CD34 is a marker for hematopoietic stem and progenitor cells. Anti-CD34 can be used in various cell biology and immunology applications, such as flow cytometry and immunohistochemistry.
Automatically generated - may contain errors
Lab products found in correlation
Anti shh, by Santa Cruz Biotechnology (3 mentions)
Image pro plus 6, by Media Cybernetics (2 mentions)
Anti gli1, by Santa Cruz Biotechnology (2 mentions)
Anti vegf, by Abcam (2 mentions)
Methanol, by Merck Group (1 mentions)
Ri 1 microscope, by Nikon (1 mentions)
Positively charged glass slides, by Thermo Fisher Scientific (1 mentions)
Anti cd80, by Abcam (1 mentions)
Anti desmin, by Santa Cruz Biotechnology (1 mentions)
Diaminobenzidine dab, by Maixin Group (1 mentions)
Qwin v3, by Leica (1 mentions)
Vascular endothelial growth factor (vegf), by Santa Cruz Biotechnology (1 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (1 mentions)
Anti ki67, by Santa Cruz Biotechnology (1 mentions)
Anti caspase 3, by Santa Cruz Biotechnology (1 mentions)
Confocal laser scanning microscope, by Olympus (1 mentions)
α sma, by Santa Cruz Biotechnology (1 mentions)
Inverted microscope, by Olympus (1 mentions)
Anti vegf, by Santa Cruz Biotechnology (1 mentions)
Eclipse 80i, by Nikon (1 mentions)
22 protocols using anti cd34
1
Angiogenesis Detection in Stroke Penumbra
2
Immunohistochemical Analysis of Knee Joints
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Briefly, 6-μm sections of knee joint tissue were placed on positively charged glass slides (Thermo Fisher Scientific, Asheville, NC, USA) and then dried with a hotplate to increase the adherence of the tissues to the slides. After the samples were deparaffinized and rehydrated according to conventional methods, endogenous peroxidases were blocked with 3% hydrogen peroxide in methanol (Sigma-Aldrich, Burlington, USA). After 30 min the samples were digested with 5 mg/ml hyaluronidase in phosphate buffered saline (PBS) (Sigma-Aldrich) for 20 min and then were incubated with anti-CD31 (1:100 dilution) (SC-56; Santa Cruz, CA, USA) and anti-CD34 (1:100 dilution) (Santa Cruz, CA, USA) antibodies overnight at 4 °C. Following treatment of the samples with appropriate biotinylated secondary antibodies, a 3,3’diaminobenzidine (DAB) streptavidin-peroxidase (SP) DAB Histostain-SP immunohistochemistry kit (ZYMED203 Laboratories/Invitrogen, Carlsbad, CA, USA) was used to visualize bound antibodies. After the sections were counterstained with hematoxylin (ZYMED Laboratories/Invitrogen), the tissues were imaged with a Nikon Ri 1 microscope (Nikon, Melville, NY, USA) (Guan et al. 2012 (link)).
3
Antibody Panel for FACS and IHC
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All antibodies used for fluorescence-activated cell sorting (FACS) or immunohistochemical analyses were obtained from BD Biosciences (San Jose, CA, USA), with the following exceptions: anti-desmin and anti-CD34 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA), anti-myosin heavy chain (MyHC; MF-20) was from the University of Iowa (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA, USA), anti-H-2Kk was from Biolegend (San Diego, CA, USA), anti-CD80 was from Abcam (Cambridge, MA, USA), and anti-CD86 was from LifeSpan BioSciences, Inc. (Seattle, WA, USA).
4
Immunohistochemical Analysis of Shh and CD34
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Sections were immersed in citrate buffer (pH 6.0) and microwaved for 20 min after a standard histochemical procedure. Then, the sections were rinsed in phosphate-buffered saline (PBS) three times for 3 min before treated with 3% hydrogen peroxide for 15 min. After blocking with PBS containing 0.3% Triton X-100 and 10% bovine serum albumin, sections were incubated with anti-Shh (1:100, Santa Cruz Biotechnology, USA) or anti-CD34 (1:100, Santa Cruz Biotechnology, USA) for 60 min at 37°C. After washing with PBS three times for 3 min, the sections were reacted with the corresponding secondary antibodies for 30 min at 37°C. The sections were incubated with fresh diaminobenzidine (DAB) (Maixin, China) for 5 min and then with enough Hematoxylin for 1 min. The stained cells were observed under a microscope (Olympus, Japan) and then counted using the Image-Pro Plus 6.0 software (Media Cybernetics, USA).
5
Immunofluorescence Staining of Tumor CD34
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Immunofluorescence staining was performed as previously described by Campolo et al. [20 (link)]. Tumor samples were collected and processed for immunofluorescence staining. Tissue sections of 7 μm were incubated with the following primary antibody anti-CD34 at 37 °C overnight (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-74499). Then, tissue sections were washed with PBS and incubated with secondary antibody anti-mouse Alexa Fluor-488 antibody (1:1000 v/v, Molecular Probes, Altrincham, UK) for 1 h at 37 °C. For nuclear staining, 4′,6′-diamidino-2-phenylindole (DAPI; Hoechst, Frankfurt, Germany) (2 μg/mL) in PBS was added. Sections were observed and photographed at 40× magnification using a Leica DM2000 microscope.
6
Immunohistochemical Localization of Key Proteins
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Immunohistochemical localization was performed as previously described by Esposito et al. [16 (link)]. Slides were incubated overnight using the following primary antibodies: VEGF (Santa Cruz Biotechnology, Dallas, TX, USA; 1:100 in PBS, v/v; sc-7269), eNOS (Santa Cruz Biotechnology, Dallas, TX, USA, 1:100 in PBS, v/v; sc-376751) anti-Bcl2 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-7382); anti-caspase-3 (1:100, Santa Cruz Biotechnology, Dallas, TX, USA; sc-56053); anti-Ki-67 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-23900); anti-CD34 (1:100; Santa Cruz Biotechnology, Dallas, TX, USA; sc-74499). At the end of the incubation with the primary antibodies, the sections were abundantly washed with PBS and incubated with a secondary antibody (Santa Cruz Biotechnology, Dallas, TX, USA) for 1 h at room temperature. The reaction was revealed by a chromogenic substrate (brown DAB), and counterstaining with NUCLEAR FAST-RED. The percentage of positive staining was measured using a computerized image analysis system (Leica QWin V3, Cambridge, UK). The images were acquired using an optical microscope (Zeiss, Axio Vision, Feldbach, Schweiz). For immunohistochemistry, the images were shown at a magnification of 20 × (50 μm of the bar scale).
7
Immunofluorescence Staining of Paraffin Sections
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Paraffin sections for IF were stained with anti-Shh (1:100, Santa Cruz Biotechnology, USA), anti-Gli1 (1:100, Santa Cruz Biotechnology, USA), anti-VEGF (1: 100, Abcam, UK) and anti-CD34 (1: 100, Santa Cruz Biotechnology, USA) overnight at 4°C. Then the sections were washed with PBS and then incubated with the secondary antibody FITC-labeled goat anti-mouse antibody (1:50, ZSGB-BIO, China) at 37°C for 1 h. Next, the cellular nuclei were stained with propidium iodide (PI) solution for 5 min in the dark. Finally, the images were captured using a confocal laser scanning microscope (Olympus, Japan) and the mean fluorescence density was analyzed by the Image-Pro Plus 6.0 software (Media Cybernetics, USA).
8
Immunostaining Analysis of HUVECs and HUASMCs
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The present study was approved by the Ethics Committee of Shanghai Jiao Tong University, Shanghai, China. The protocol was performed according to the protocol used in our previous study (Zhang et al. 2011 (link)).
Immunohistochemistry and immunofluorescence studies were carried out for HUVECs and HUASMCs by using the primary antibodies against CD34 (anti-CD34; dilution, 1:100; Santa Cruz), VEGF (anti-VEGF; dilution, 1:50; Santa Cruz), factor VIII (anti-factor VIII; dilution, 1:50; Santa Cruz) and α-SMA (anti-smooth muscle α-actin; dilution, 1:200; Santa Cruz). The immunostained cells were then visualised under an inverted microscope (Olympus).
Immunohistochemistry and immunofluorescence studies were carried out for HUVECs and HUASMCs by using the primary antibodies against CD34 (anti-CD34; dilution, 1:100; Santa Cruz), VEGF (anti-VEGF; dilution, 1:50; Santa Cruz), factor VIII (anti-factor VIII; dilution, 1:50; Santa Cruz) and α-SMA (anti-smooth muscle α-actin; dilution, 1:200; Santa Cruz). The immunostained cells were then visualised under an inverted microscope (Olympus).
9
Immunohistochemical Analysis of ELTD1 Expression
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Paraffin-embedded samples were cut into 4-μm sections and processed for IHC. Tissue sections prepared for antigen retrieval by microwave treatment in citrate buffer (pH 6.0) were incubated with anti-ELTD1 (Proteintech Group, Chicago, IL, USA), anti-αSMA (Cell Signaling Technology, Danvers, MA, USA), anti-vimentin (Cell Signaling Technology), and anti-CD34 (Santa Cruz, Dallas, Texas, USA) primary antibodies. Immunostaining was performed using the Envision System with diaminobenzidine (Dako Cytomation, Glostrup, Denmark). Images were viewed and assessed using a microscope (Eclipse 80i, Nikon, Tokyo, Japan). Assessments of the ELTD1 staining were scored by two experienced pathologists blinded to patient identity and clinical status. The staining analysis technique was performed as previously described 24 (link). In particular, the proportion score was assigned based on the proportion of positive cells and the intensity of staining. The proportion of positive cells was scored from 0% to 100%. The intensity of staining was scored from 0 to 3 (0, none; 1, weak; 2, intermediate; 3, strong). The final quantitation of each staining was obtained by multiplying the two scores. For patient survival analyses, the median was set as the cut-off to distinguish strong and weak staining of ELTD1.
10
Investigating IMD1-53 Peptide Effects
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IMD1–53 peptide was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, CA, USA). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA USA). Phosphorylated (p)-AMPKαThr-172 (cat. no. 2535), AMPKα (cat. no. 2532), p-AktSer-473 (cat. no. 4060), and p-endothelial nitric oxide synthase (p-eNOS)Ser-1179 (cat. no. 9570) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Anti-Akt (cat. no. sc-8312), anti-eNOS (cat. no. sc-654), anti-GAPDH (cat. no. sc-25778) and anti-VEGF (cat. no. sc-152), anti-CD31 (cat. no. sc-1505) anti-CD34 (cat. no. sc-7045) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Compound C (Comp C) was purchased from Toronto Research Chemicals Inc. (North York, ON, Canada). Goat anti-rabbit (cat. no. sc-2030) and goat anti-mouse (cat. no. sc-3791) secondary antibodies were purchased from Santa Cruz Biotechnology, Inc.
For all experiments concerning IMD1–53, the same volume of PBS was used as a vehicle. For experiments concerning Comp C, the same volume of DMSO was used as a vehicle.
For all experiments concerning IMD1–53, the same volume of PBS was used as a vehicle. For experiments concerning Comp C, the same volume of DMSO was used as a vehicle.
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