The blood vessels amount of tumor sections were stained by CD31 immunofluorescence and CD34 immunohistochemical to assess the tumor suppression effect. The corresponding expressions CD8 in tumor sections were analyzed by immunohistochemical staining to evaluate the regulatory effect of each treatment group on immune-related factors. For immunohistochemical staining, the tumor slices were treated with different primary antibodies according to the protocols, including CD31 antibody (Abcam, Cambridge, UK), CD34 antibody (Abcam, Cambridge, UK), and CD8 antibody (Abcam, Cambridge, UK). Three regions of each section were chosen randomly and quantitatively analyzed by Image-Pro Plus software. The expression of the target protein in tumor tissues was quantitatively evaluated by WB.
Cd34 antibody
Manufactured by Abcam
Sourced in United States, United Kingdom
The CD34 antibody is a laboratory reagent used to detect the presence of the CD34 protein, which is a cell surface marker. It is commonly used in immunohistochemistry and flow cytometry applications for the identification and characterization of hematopoietic stem and progenitor cells.
Automatically generated - may contain errors
Lab products found in correlation
Cd31 antibody, by Abcam (2 mentions)
Na 0.8 plan apochromat objective, by Zeiss (2 mentions)
Target retrieval solution, by Agilent Technologies (2 mentions)
Axio scan z1 microscope, by Zeiss (2 mentions)
Cd8 antibody, by Abcam (1 mentions)
P h2ax ser139, by Merck Group (1 mentions)
P akt, by Cell Signaling Technology (1 mentions)
Vectastatin abc kit, by Vector Laboratories (1 mentions)
Pecam 1, by Santa Cruz Biotechnology (1 mentions)
Normal goat serum (ngs), by Agilent Technologies (1 mentions)
Gfap antibody, by Abcam (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Cleaved caspase 3 antibody, by Abcam (1 mentions)
Hematoxylin, by Maixin Group (1 mentions)
Elivision plus, by Maixin Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Msc medium, by ScienCell (1 mentions)
Vwf antibody, by Abcam (1 mentions)
Fitc conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Cdna synthesis kit, by Bio-Rad (1 mentions)
10 protocols using cd34 antibody
1
Tumor Vasculature and Immune Response
2
PANC-1 Xenograft Tumor Immunohistochemistry
3
Immunohistochemical Analysis of Angiogenic Markers
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After incubation in 3% H2O2 followed by blocking in 10% normal goat serum (Dako Corporation, Carpentaria, CA) in PBS, the sections were immunostained overnight at 4°C with antibodies against von Willebrand factor (vWF, dilution 1∶200; Dako), platelet endothelial cell adhesion molecule-1 (PECAM-1; dilution 1∶50, Santa Cruz Biotechnology, Santa Cruz, CA), proliferating cell nuclear antigen (PCNA; dilution 1∶200, Dako), CD34 antibody (dilution 1∶100; Abcam, Cambridge, MA). The sections were then treated with secondary antibodies (Vector Laboratories, Burlingame, CA). Immunoreactivity was visualized using the avidin-biotin complex method (Vectastatin ABC kit, Vector Laboratories) and developed with diaminobenzidine, followed by counterstaining with Gill hematoxylin (Sigma-Aldrich, St. Louis, MO).
4
Histological Analysis of Mouse Bone and Vasculature
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Long bones were decalcified in 10% EDTA, embedded in paraffin and sections were stained with hematoxylin, eosin (H&E) and orange G. All mouse brains were stained with H&E. For IHC, slides were autoclaved at 121°C for 10 min in target retrieval solution (DAKO, Santa Clara, CA, USA), followed by CD34 antibody (1:2 000, Abcam). Tartrate-resistant acid phosphatase (TRAP) staining was performed as previously described (18 (link)). For histomorphometric analysis, bright-field microscopic images were acquired randomly with a AxioScan Z1 microscope using a 20X/NA 0.8 plan apochromat objective (Zeiss, Oberkochen, Germany). CD34-stained blood vessel areas were quantified using ImageJ (NIH, Bethesda, MD, USA) as described previously (34 (link)). TRAP+ osteoclast number per millimeter of tumor/bone interface was counted at 20X magnification as described previously (34 (link), 54 (link)). All the analysis was performed blindly.
5
Immunofluorescence Assay for Neural Cell Analysis
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Immunofluorescence analysis was performed as previously described (Jing et al., 2015 (link); Xiong et al., 2017 (link)). Briefly, sections were processed for immunofluorescent labeling with CD34 antibody (for endothelial cells of microvessels), glial fibrillary acidic protein (GFAP) antibody (for astrocytes), NeuN antibody (for neurons) and cleaved caspase-3 (C-caspase 3) antibody (for apoptotic cells). The sections were immersed in blocking solution (5% normal goat serum or donkey serum in PBS) at 20°C for 2 h, then incubated 12 h at 4°C with CD34 antibody (rabbit, 1:100; Abcam, United States), GFAP antibody (rabbit, 1:1000; Abcam, United States),and NeuN antibody (mouse, 1:100; Abcam, United States) and cleaved caspase-3 antibody (rabbit, 1:100; Abcam, United States). After three times of washing with 0.01 M PBS, the sections were incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit IgG, 1:400; Jackson Immunoresearch, West Grove, PA, United States). CY3- conjugated goat anti-rabbit or goat anti-mouse IgG, 1:300; Servicebio,China) for 2 h at 20°C. After three times of washing in PBS, the sections were covered with anti-quenching fluorescence mounting medium.
6
Sericin-Modified PLLA Endothelialization Membrane
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A CD34 antibody (Abcam, Cambridge, UK) solution with a concentration of 4 μg/mL in sterile PBS (pH = 7.4) was prepared. The sericin-modified PLLA membrane was soaked in the CD34 antibody solution and incubated overnight to obtain the modified PLLA endothelialization membrane.
7
Quantifying Tumor Angiogenesis via CD34 IHC
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Sections cut from paraffin-embedded nude mice tumor samples were deparaffinized and rehydrated. Immunohistochemical staining assay was used to detected the expression of CD34 in all tumor sections with CD34 antibody (1:250, Abcam, USA) and Elivision™ plus (Maixin Biotechnology Co. Ltd, China). Then cell nucleus was stained with hematoxylin (Maixin Biotechnology Co. Ltd, China). After hydration and transparent, sections were sealed with neutral resins and photographed. Finally, CD34 positive vessels which were defined as blood vessels were analyzed with Image Pro Plus.
8
Histological Analysis of Mouse Bone and Vasculature
9
Hydrogel-based MSC Culture and Characterization
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G1-CS hydrogel (prepared as described in a previous report (Zhang et al., 2019a )), MSC medium (ScienCell Research Laboratories, Carlsbad, CA, USA), fetal bovine serum (Gibco Life Technologies, Carlsbad, CA, USA), penicillin-streptomycin (Gibco, 15140122), TRIzol (Invitrogen, Carlsbad, CA, USA), SYBR Green Supermix (Bio-Rad, Hercules, CA, USA), cDNA synthesis kit (Bio-Rad), 4 % paraformaldehyde (Servicebio, Wuhan, China), CD31 antibody (Abcam, Cambridge, UK), vWF antibody (Abcam), CD34 antibody (Abcam), CD90 antibody (Abcam), FLK-1 antibody (Abcam), GIT1 antibody (Abcam), and GAPDH antibody (Abcam). Secondary antibody (Abcam), FITC-conjugated secondary antibody (Jackson ImmunoResearch, West Grove, PA), DAPI (Beyotime Institute of Biotechnology, Jiangsu, China), Alkaline Phosphatase staining kit (Servicebio), PBS (Gibco), Opti-MEM medium (Gibco), AAV-GIT1 and Plasmids-GIT1 [constructed by Obio Technology (Shanghai) Company, Shanghai, China], BD Matrigel (BD Bioscience, San Jose, CA, USA), and Microfil MV-122 (Flow Tech Inc., Carver, MA, USA).
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10
Matrigel Plug Angiogenesis Assay
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Matrigel plug assay was performed as described by Malinda (2003) . 0.5 mL Matrigel containing 50 µL CM (concentration 50×, MenSCs group) or ECM (control group) or PBS (negative group) were injected subcutaneously into the ventral area of C57/BL/6 mice (n = 5 per group). After 7 days, the mice were killed and intact Matrigel plugs from three groups of mice were removed. The removed Matrigel plugs were photographed and were fixed with an optimal cutting temperature (OCT) compound, and 6 µm-thick fresh sections were made for IHC staining to identify the migration of the endothelial cells and infiltration of new microvessels. Slides were washed twice with PBS and incubated with 5% bovine serum albumin (BSA) for 30 min at room temperature. Sections were incubated overnight at 4°C with CD34 antibody (Abcam). At least three random fields of the area underlying the skin of each mouse were counted.
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