IHC was performed according to the protocol of anti-LRG1 antibody (Abcam, MA, USA) and anti-CD34 antibody (Abcam, MA, USA). Briefly, paraffin sections were first dewaxed and then hydrated. After microwave-based antigen retrieval with 10 mM citrate buffer (pH 6.0), endogenous peroxidase activity was blocked with incubation of the slides in 3% H2O2, and non-specific binding sites were blocked with 10% goat serum. After blocking, the slides were incubated with primary anti-LRG1 antibody (1:150 dilution) and anti-CD34 antibody (1:150 dilution) overnight in a moist chamber at 4°C. Slides were washed in PBS for three times, before being incubated with HRP-conjugated secondary antibody for 0.5 hour. Then the slides were stained with the DAKO Liquid 3,’3-diaminobenzidine tetrahydrochloride (DAB). Finally, the slides were counter stained with hematoxylin and observed under microscope. Negative control slides omitting the primary antibodies were included in all assays.
Anti cd34 antibody
Manufactured by Abcam
Sourced in United States, China, United Kingdom
Anti-CD34 antibody is a laboratory reagent used for the detection and analysis of CD34, a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells. This antibody can be used in various immunological techniques, such as flow cytometry, immunohistochemistry, and western blotting, to identify and characterize CD34-positive cell populations.
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Lab products found in correlation
Anti lrg1 antibody, by Abcam (1 mentions)
Anti ki67 antibody, by ZSGB-BIO (1 mentions)
Anti pcna antibody, by Abcam (1 mentions)
Tunel assay, by Roche (1 mentions)
F4 80 antibody, by Abcam (1 mentions)
Pimonidazole hydrochloride, by Merck Group (1 mentions)
Avidin biotin peroxidase complex, by Agilent Technologies (1 mentions)
3 3 diaminobenzidine (dab), by Merck Group (1 mentions)
Bx41 microscopic imaging system, by Olympus (1 mentions)
Anti brdu antibody, by Abcam (1 mentions)
Fluorescent microscope, by Nikon (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Beyotime (1 mentions)
Fitc labeled igg, by Santa Cruz Biotechnology (1 mentions)
Rhodamine tritc labeled igg, by Thermo Fisher Scientific (1 mentions)
Dmi3000 microscope, by Leica camera (1 mentions)
Anti cytokeratin 18, by Boster Bio (1 mentions)
Anti vimentin, by Cell Signaling Technology (1 mentions)
Anti acsl4 antibody, by Abcam (1 mentions)
Anti a20 antibody, by Abcam (1 mentions)
Biotinylated secondary antibody, by Boster Bio (1 mentions)
18 protocols using anti cd34 antibody
1
Immunohistochemical Analysis of LRG1 and CD34 Markers
2
Immunohistochemical Analysis of Glioma Tumor Markers
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The whole brains of the mice were harvested on day 28 after the allogeneic glioma cells were implanted, fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 15 μm thick coronal slices. The sections with the largest tumor area were stained with hematoxylin and eosin or were used for IHC staining.
For IHC analysis, sections were incubated with primary antibodies [1:100 dilution; anti-PPRA antibodies were purchased from Cell Signaling Technology (Proteintech), the anti-Ki67 antibody was purchased from Zsgb Bio (Beijing, China) and the anti-CD34 antibody was purchased from Abcam] overnight at 4 °C, followed by a 1 h incubation at 37 °C with a biotinylated secondary antibody (1:100 dilution). The samples were then incubated with horseradish peroxidase-labeled streptomycoidin and DAB (diaminobenzidine), counterstained with hematoxylin, and visualized using a light microscope.
For the IHC analysis, we quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. We assigned the following proportion scores: 1 if 0–25% of the tumor cells showed positive staining, 2 if 26–50% of cells were stained, 3 if 51–75% of the cells were stained, and 4 if 76–100% of the cells were stained; we also divided the different expression levels into four different groups (1 to 4) and scored them.
For IHC analysis, sections were incubated with primary antibodies [1:100 dilution; anti-PPRA antibodies were purchased from Cell Signaling Technology (Proteintech), the anti-Ki67 antibody was purchased from Zsgb Bio (Beijing, China) and the anti-CD34 antibody was purchased from Abcam] overnight at 4 °C, followed by a 1 h incubation at 37 °C with a biotinylated secondary antibody (1:100 dilution). The samples were then incubated with horseradish peroxidase-labeled streptomycoidin and DAB (diaminobenzidine), counterstained with hematoxylin, and visualized using a light microscope.
For the IHC analysis, we quantitatively scored the tissue sections according to the percentage of positive cells and staining intensity. We assigned the following proportion scores: 1 if 0–25% of the tumor cells showed positive staining, 2 if 26–50% of cells were stained, 3 if 51–75% of the cells were stained, and 4 if 76–100% of the cells were stained; we also divided the different expression levels into four different groups (1 to 4) and scored them.
3
Evaluation of Renal Tissue Integrity and Cellular Responses
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To assess the morphological integrity, the harvested tissues at 24 hours after reperfusion were fixed in paraffin, sectioned and analysed by haematoxylin and eosin staining (H&E). Histological score of the kidney (HSK) was evaluated in a blind manner by two experienced pathologists. As described previously,2 three fields per section were scored on a scale of 0‐4 (0, 0%; 1, 0%‐5%; 2, 5%‐25%; 3, 25%‐75%; and 4, 75%‐100%). The effect of LESW on apoptosis, cell proliferation and small vascular density was observed at 3 days after reperfusion. Apoptosis was evaluated with terminal transferase–mediated deoxyuridine triphosphate nick‐end‐labelling (TUNEL) assay (Roche) according to the manufacturer's protocol. Proliferation of renal cell was detected by using antiproliferating cell nuclear antigen (anti‐PCNA) antibody (Abcam). Small vascular density was measured by anti‐CD34 antibody (Abcam). Anti‐SDF‐1 antibody (Abcam) was used to observe the cell types that express SDF‐1 at 24 hours after reperfusion. Immunohistochemical assays for the staining of anti‐PCNA, anti‐CD34 and anti‐SDF‐1 were performed according to our previous protocol.23
4
Tumor Targeting of Functionalized Nanoparticles
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MPNPs or M1-MPNPs were intravenously injected into 4T1 tumor-bearing BALB/c mice at an equivalent MTPE-TT dose. The fluorescence imaging of animals was observed by IVIS (Berthold Technologies, NightOWL II LB983, Germany) at 2 h, 8 h, 12 h, 24 h, 36 h, and 48 h post NPs injection. The semiquantitative fluorescence intensity of tumor sites from the in vivo images were also quantified. After 48 h of injection, organs were excised from the sacrificed mice for imaging. Immunofluorescence staining was also used to investigate the tumor accumulation and distribution of MPNPs or M1-MPNPs. The 5 μm thickness of tumor sections were cut and stained with anti-CD34 antibody (1:200, Abcam, Rabbit mAb, #ab81289) and DAPI, and then imaged with CLSM.
5
Skin Angiogenesis and Macrophage Quantification
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Capillary density in skin tissue was assessed morphometrically by examining four random fields of equal size per section of wound excluding wound edges in six successive sections after immunofluorescence staining for endothelial cells with an anti-CD34 antibody (1:100, Abcam, Cambridge, MA, USA). Macrophages were identified by staining with an F4/80 antibody (1:100, Abcam, Cambridge, MA, USA). For immunofluorescence staining, tissue sections were treated with antigen retrieval solution at 95 °C for 10 min after rehydration and then with 3% hydrogen peroxide to inactivate endogenous peroxidases. Tissue sections were incubated with the primary antibody for 24 h at 4 °C and then with horseradish peroxidase-conjugated secondary antibody at 37 °C for 45 min. Sections were washed, and signals were observed under a fluorescence microscope.
6
Tumor Hypoxia and Angiogenesis Assessment
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Immunohistochemical staining with CD34 or pimonidazole was performed on formalin-fixed and paraffin-embedded tissue sections. Briefly, after antigen retrieval, tissue sections were incubated with anti-CD34 antibody (Abcam, USA) overnight at 4°C, and then incubated with biotinylated secondary antibody, and then with avidin–biotin–peroxidase complex (DAKO, Glostrup, Denmark). Finally, the tissue sections were incubated with 3′, 3′-diaminobenzidine (Sigma, St Louis, MO, USA) and counterstained with hematoxylin. In negative controls, primary antibodies were not applied. Pimonidazole hydrochloride (Chemicon International, Temecula, CA, USA) was used to detect the tumor hypoxia as previously described.23 (link) Briefly, tumor-bearing mice were intraperitoneally injected with Pimonidazole hydrochloride (0.1 mg/g body weight) dissolved in 10 mg/ml in 0.9% saline 1 h before sacrifice. The ratio of pimonidazole-positive area (%) was defined as the pimonidazole-positive area divided by the visible tumor area under 100-fold magnifications. The tumor micro-vessel density (MVD) was expressed as the ratio of CD34 positive stained area per total tumor area in a 200× high-power field.
7
Immunofluorescence Staining of Frozen Tissues
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Immunostaining was performed with frozen tissue sections as previously described. Tissues were fixed in methanol at -20°C for 20 min. After blocking with PBS with 3% FBS, tissues were incubated with anti-BrdU antibody (1:50, abcam, USA) and/or anti-CD34 antibody (1:10, abcam, USA) for 60 min at room temperature. Fluorescently labelled anti-rabbit (1:200, abcam, USA) or anti-goat antibody (1:200, abcam, USA) was incubated with the tissues for 45 min. After washing four times with PBS, the slides were mounted with Mountshield that contained with DAPI. Fluorescent images were captured and processed using the Olympus BX41 microscopic imaging system (Center Valley, PA, USA).
8
Immunofluorescent Staining of Cultured EPCs
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After 7 days in culture, attached EPCs were fixed by 4% paraformaldehyde for 10 min at room temperature (RT). After washing with PBS three times, cells were stained with anti-CD31 antibody (cat. no. 550274; 1:100; BD Biosciences) or anti-CD34 antibody (cat. no. ab81289; 1:100; Abcam) at 4°C overnight. Next, the cells were incubated with Dylight 488-conjugated goat anti-rat secondary antibody (cat. no. A23220; 1:500; Abbkine Scientific, Co., Ltd.) or Dylight 594-conjugated goat anti-rabbit secondary antibody (cat. no. A23420; 1:500; Abbkine Scientific, Co., Ltd.) for 1 h at RT. Nuclei were counterstained with DAPI (10 µg/ml; Beyotime Institute of Biotechnology), and cells were examined with a fluorescent microscope (Nikon Corporation).
9
Immunofluorescence Analysis of Cultured hAECs
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hAECs were fixed with 4% paraformaldehyde for 20 minutes at room temperature and then washed twice (5 minutes each) with PBS. Cells were permeabilized with 0.1% Triton X-100 for 10 minutes at room temperature and then washed twice with 1 × PBS. Then cells were blocked with blocking solution for 30 minutes and incubated overnight at 4°C with anti-Cytokeratin 18 (mouse anti-human 1 : 200, Boster, Wuhan, China), anti-CD34 antibody (rabbit anti-human 1 : 100, Abcam, Cambridge, MA, USA), and anti-Vimentin (rabbit anti-human 1 : 100, Cell Signaling Technology, Danvers, MA, USA). Washed three times with PBS, the cells were probed with FITC-labeled IgG (1 : 200, Santa Cruz, CA, USA) or Rhodamine- (TRITC-) labeled IgG (1 : 100, Invitrogen, CA, USA). Fluorescence images were obtained with a Leica DMI3000 microscope (Heidelberg, Germany).
10
Immunohistochemical Analysis of Tumor Tissues
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Animal tumor tissues were sectioned and stained with a 1:1000 dilution of anti-ACSL4 antibody (Abcam), 1:1000 dilution of anti-A20 antibody (Abcam), 1:500 dilution of anti-CD34 antibody (Abcam). Five regions were selected randomly for each specimen.
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