The VEGF-D expression and lymphatic vessels of GBC specimens were detected by immunohistochemistry as previously described [21 (link)]. The primary antibodies were VEGF-D (ab155288, Abcam) at a 1:80 dilution and LYVE-1 (AF2125, R&D Systems) at a 1:150 dilution. The method used to measure the VEGF-D expression has been described previously [23 (link)]. The density of LYVE-1-positive vessels (lymphatic vessels density, LVD) was assessed according to the method described by Qiang Du [24 (link)].
Ab155288
Manufactured by Abcam
Sourced in United Kingdom
Ab155288 is a laboratory equipment product. It is a device designed for scientific research and analysis purposes. The core function of this product is to facilitate specific tasks and procedures within a laboratory setting. However, a detailed description of its features and intended use is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Af2125, by R&D Systems (1 mentions)
Rnase inhibitor, by Takara Bio (1 mentions)
Igg1 pe, by BioLegend (1 mentions)
Oligo dt 15 primer, by Takara Bio (1 mentions)
Dmso d2650, by Merck Group (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Matrigel, by BD (1 mentions)
Ab86607, by Abcam (1 mentions)
Ab51074, by Abcam (1 mentions)
Ab76003, by Abcam (1 mentions)
Ab180630, by Abcam (1 mentions)
Ecl detection system, by Merck Group (1 mentions)
Ab185696, by Abcam (1 mentions)
E ab 20058, by Elabscience (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Mab3300, by Merck Group (1 mentions)
Ab46154, by Abcam (1 mentions)
Ab32152, by Abcam (1 mentions)
5 protocols using ab155288
1
Immunohistochemical Analysis of VEGF-D and Lymphatic Vessels in Gallbladder Carcinoma
2
Comprehensive Cell Culture Methodology
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RPMI1640 (C11875500BT), DMEM/F12 (11330032), fetal bovine serum (10099141), Trypsin (25200072), and antibiotic-Antimycotic (15240062) were purchased from Gibco BRL (Grand Island, NY, USA). A fluorescein isothiocyanate (FITC) BrdU flow kit (559619), Annexin-V-FITC Apoptosis Detection kit (556547), and Matrigel (356230) were purchased from BD Biosciences (San Jose, CA, USA), and TRIzol (15596026) was purchased from Invitrogen (Grand Island, NY, USA). An ELISA kit to detect the secretion of VEGF-D (ELH-VEGFD-001) was purchased from RayBiotech (Norcross, GA, USA), and an ELISA kit to detect secretion of VASH2 (ab155288) was purchased from Abcam (Cambridge, UK). DMSO (D2650) was purchased from Sigma (St. Louis, MO, USA), and Oligo-(dT)15 primer, 10-mM dNTP, RNase inhibitor, and an SYBR Premix Ex TaqTM kit (RR820A) were purchased from Takara (Tokyo, Japan). In the flow cytometry assay, all fluoresce-labeled antibodies were purchased from Biolegend (San Diego, CA, USA), including phycoerythrin (PE) anti-human podoplanin lymphatic endothelial cell surface markers, and PE-IgG1. In immunohistochemistry, all antibodies were purchased from Bioss (Beijing, China), and biotinylated secondary goat anti-mouse or rabbit IgG antibody were purchased from Santa Cruz (Dallas, TX, USA).
3
Quantification of Angiogenesis-Related Proteins
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The HaCat cells cultured with or without naringin were harvested and total cell protein was extracted using whole cell lysis buffer. The protein concentrations were determined by the Bradford method (Bio-Rad, CA, USA). Samples with an equal amount of protein were subjected to 8–15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a polyvinylidene difluoride (PVDF) (Millipore, Bedford, MA, USA) membrane. The membrane was incubated at room temperature in blocking solution (5% nonfat milk) for 1 h followed by incubation for 2 h in blocking solution containing an appropriate dilution of anti-MMP2 (ab86607, abcam), MMP-9 (ab76003, abcam), MMP-14 (ab51074, abcam), TIMP-1 (MAB3300, millipore), TIMP-2 (ab180630, abcam), VEGF-A (ab46154, abcam), VEGF-B(ab185696, abcam), VEGF-C (ab191274, abcam), VEGF-D (ab155288, abcam), VEGF-R1(ab32152, abcam), VEGF-R2 (9698, cell Signaling), VEGF-R3 (2485, cell Signaling), and β actin (E-AB-20058, Elabscience). After washing, blots were then probed with appropriate secondary horseradish peroxidase (HRP)-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) and detected by an ECL detection system (Millipore). β-actin served as internal control.
4
Immunocytochemical Analysis of Stem Cell Secretome
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DPSCs and SHED were cultured on coverslips coated with poly-L-lysin. After washing with PBS (Invitrogen), the samples were fixed with 4% paraformaldehyde. The fixed cells were permeabilized with PBS containing 0.25% Triton X-100 (PBST; Bio Basic, Seoul, Korea), washed, and incubated with 5% BSA (Sigma–Aldrich) in PBST to block nonspecific antibody binding.
The cells were incubated in primary antibodies diluted in 5% BSA/PBST overnight at 4 °C. The primary antibodies included antihuman interleukin-6 (IL-6; rabbit polyclonal antibody, ab6672, Abcam; 1:500), antihuman brain-derived neurotrophic factor (BDNF; rabbit monoclonal antibody, ab108319, Abcam; 1:200), antihuman placental growth factor (PLGF; rabbit polyclonal antibody, ab9542, Abcam; 1:500), and antihuman vascular endothelial growth factor D (VEGF-D; rabbit monoclonal antibody, ab155288, Abcam; 1:200). After washing, the cells were incubated with biotinylated secondary antibody (biotinylated antirabbit IgG, Vector Labs, Burlingame, CA, USA) in 5% BSA/PBST for 1 h at RT. After washing, the cells were incubated with streptavidin–HRP (Vector Labs) in 5% BSA/PBST for 30 min at RT. Color was developed using 3,3ʹ-diaminobenzidine substrate (Abcam) and hematoxylin (Merck). Coverslips were mounted using a drop of mounting medium (Vector Labs) and stored at RT.
The cells were incubated in primary antibodies diluted in 5% BSA/PBST overnight at 4 °C. The primary antibodies included antihuman interleukin-6 (IL-6; rabbit polyclonal antibody, ab6672, Abcam; 1:500), antihuman brain-derived neurotrophic factor (BDNF; rabbit monoclonal antibody, ab108319, Abcam; 1:200), antihuman placental growth factor (PLGF; rabbit polyclonal antibody, ab9542, Abcam; 1:500), and antihuman vascular endothelial growth factor D (VEGF-D; rabbit monoclonal antibody, ab155288, Abcam; 1:200). After washing, the cells were incubated with biotinylated secondary antibody (biotinylated antirabbit IgG, Vector Labs, Burlingame, CA, USA) in 5% BSA/PBST for 1 h at RT. After washing, the cells were incubated with streptavidin–HRP (Vector Labs) in 5% BSA/PBST for 30 min at RT. Color was developed using 3,3ʹ-diaminobenzidine substrate (Abcam) and hematoxylin (Merck). Coverslips were mounted using a drop of mounting medium (Vector Labs) and stored at RT.
5
Cryosectioning and Immunofluorescence of Tumor Samples
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Cryomold tumour samples were cryosectioned in 5 µm‐thick sections and immediately mounted on slides. The sections were post‐fixed with 4% paraformaldehyde and stained either with haematoxylin and eosin (HE) or for immunofluorescence (IF), as described.23 Tissue sections were heated for 10 min in 10 mM sodium citrate pH 6.0 for antigen retrieval. Sections were permeabilized for 5 min in 0.3% Triton X‐100 PBS solution before blocking for 1 h in 0.3% milk,10% bovine serum albumin and 0.3% Triton X‐100 in PBS. Primary antibodies were monoclonal rabbit anti‐VEGFD (ab155288; Abcam) and monoclonal mouse anti‐PDGFRB.24 Secondary antibodies were donkey anti‐rabbit Alexa Fluor 594 (Invitrogen # A‐21207) and donkey anti‐mouse Alexa Fluor 488 (Invitrogen # A‐21202). Primary and secondary antibodies were diluted in blocking solution and incubated at 4°C overnight and 37°C for 2 hours, respectively. Hoechst (Invitrogen) was used to stain nuclei. Fluorescence was observed with a Zeiss Axiovert 200 inverted fluorescence microscope (Zeiss). HE slides were scanned using an Oyster imaging system (3DHistech).
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