Cells were homogenized in either Radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher) supplemented with protease inhibitor mixture (Pierce), or directly in 1× Laemmli buffer (Bio-rad, Hercules, CA, USA) supplemented with β-mercaptoethanol (Sigma-Aldrich, St. Louis, MO, USA). Protein concentrations of RIPA lysates were determined using a bicinchoninic acid (BCA) assay kit (Thermo Fisher) with BSA as a standard. Proteins (20–40 μg) were separated on a run on 4-20% polyacrylamide Tris-glycine gels (Thermo Fisher) and transferred to polyvinylidene difluoride (PVDF) membranes. The transferred membranes were blocked for 1 hour at room temperature in Odyssey Blocking Buffer (LI-COR, Lincoln, NE, USA) and incubated at 4°C overnight with primary antibodies against: human and rhesus PECAM1 (1:500; JC/70A, Abcam, Cambridge, MA, USA), human and mouse VE-cadherin (1:200, C-19, Santa Cruz Biotechnologies, Santa Cruz, CA, USA), phospho-AKT (Ser473) (1:1000, 12694S, Cell Signaling, Danvers, MA, USA), phospho-ERK1/2 (Thr202/Tyr204) (1:1000, 4370, Cell Signaling), α-tubulin (1:5000, ab89984, Abcam), Vinculin (1:1000, V4139, Sigma), and β-actin (1:1000, ab13822, Abcam). The signals were visualized with an Odyssey imaging system.
Jc 70a
Manufactured by Abcam
Sourced in United States, United Kingdom
The JC/70A is a monoclonal antibody that recognizes the CD31 antigen, also known as PECAM-1. CD31 is a cell adhesion molecule expressed on the surface of endothelial cells, platelets, and certain leukocyte subsets. The JC/70A antibody can be used to identify and characterize these cell types.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey blocking buffer, by LI COR (1 mentions)
Laemmli buffer, by Bio-Rad (1 mentions)
Radio immunoprecipitation assay ripa buffer, by Thermo Fisher Scientific (1 mentions)
Ab13822, by Abcam (1 mentions)
Ab89984, by Abcam (1 mentions)
β mercaptoethanol, by Merck Group (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
V4139, by Merck Group (1 mentions)
Prolong gold antifade mountant, by Thermo Fisher Scientific (1 mentions)
Alexa fluor 546 conjugated goat anti mouse igg, by Thermo Fisher Scientific (1 mentions)
Nanozoomer 2.0 rs, by Hamamatsu Photonics (1 mentions)
Duel endogenous enzyme block, by Agilent Technologies (1 mentions)
Alexa fluor 488 goat anti mouse igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Dm6b microscope, by Leica (1 mentions)
Paraformaldehyde (pfa), by Wuhan Servicebio Technology (1 mentions)
Ab7817, by Abcam (1 mentions)
Spectramax id5 microplate reader, by Molecular Devices (1 mentions)
Clone mib 1, by Agilent Technologies (1 mentions)
Ab199, by Abcam (1 mentions)
Axio scope a1 microscope, by Zeiss (1 mentions)
10 protocols using jc 70a
1
Western Blot Protein Analysis Protocol
2
Quantifying Cell-Cell Interactions via Confocal Microscopy
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We performed a series of confocal microscopy image acquisition on 16-h co-cultured cells. Cells were fixed with a 10% formalin solution neutrally buffered (Sigma-Aldrich, St. Louis, MO, USA) at RT for 15 min and were permeabilized with 0.1% Triton X-100 in PBS for 15 min. Cells were then washed two times with PBS and were blocked in 0.5% bovine serum albumin (BSA) in PBS for 20 min before incubating overnight with the primary mouse monoclonal anti-CD31 antibody (1:500 clone JC/70A, AbCam, Cambridge, UK) and a mouse monoclonal anti-CD42b-FITC antibody (1:500; eBioscience, San Diego, CA, USA). The secondary antibody for the anti-CD31 was Alexa Fluor 546-conjugated goat anti-mouse IgG (1:1,000; Thermo Fisher Scientific, Waltham, MA, USA). About 4′,6-diaminophenyl-indole (DAPI) in ProLong® Gold Antifade Mountant (Thermo-Fisher, Waltham, MA, USA) was used for nucleic acid staining. Confocal images were obtained with a Nikon A1 MP confocal scanning system connected to an Eclipse T-i microscope, with an × 40 objective plus further 1 and × 3 magnification, acquired by Nis-Elements imaging software, and were analyzed by the processing Image-J/Fiji software (LOCI, University of Wisconsin-Madison, USA). The degree of co-localization was quantified using Mander's overlap coefficient (MOC). Data are presented as mean ± SEM with respect to untreated samples, from a minimum of 50 cells.
3
Multiplex Immunohistochemical Analysis of Tumor Microenvironment
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Tissue samples were fixed for at least 48 h in 10% neutral buffered formalin, processed routinely, embedded in paraffin, sectioned 3 μm thick, mounted on glass slides, and stained with hematoxylin and eosin (HE). All tumors were examined for KDM2B expression and the presence of lymphocytes and macrophages using immunohistochemistry (IHC). IHC was performed using antibodies to Iba1 (1:2000, Fujifilm Wako, Osaka, Japan, 019-19741), CD3 (1:1000; Agilent Technologies, CA, USA, IR503), KDM2B (1:50; Santa Cruz Biotechnology, Inc.,sc-293279), CD31 (1:250; Abcam, JC/70A), PD-L1 (1:100; clone 6C11-3A11), and CD204 (1:800; Medicinal Chemistry Pharmaceutical Co., Ltd., Sapporo, Japan, KT022) as described previously7 (link). Nano Zoomer 2.0-RS (Hamamatsu Photonics, Hamamatsu, Japan) was used to scan histological slides, which were then processed in QuPath ver 0.2.134 (link). Scanned slides were opened in QuPath as Brightfield (H-DAB), and the Estimate Stain Vectors feature was used to automatically adjust the staining colors. Normal endothelial and tumor cells were detected using the Cell Detection function. The Create Detection Classifiers function was used to label cells based on their morphologies and locations, allowing the QuPath software to correctly classify each cell type. The collected data was exported and used for further analysis.
4
Immunohistochemistry of SARS-CoV-2 Spike Protein
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The tissue slides were deparaffinized, rehydrated, and blocked with Duel Endogenous Enzyme Block (Dako). Tissue sections were probed with the primary antibody against fibrin (59D8; Millipore), CD42b (SP219; Abcam; RRID: AB_2814749), VWF (F8/86; Invitrogen; RRID: AB_11001165), PECAM-1 (JC/70A; Abcam; RRID: AB_307284), or SARS-CoV-2 spike S1 protein (007; Sino Biological; AB_2827979) and then treated with HRP-conjugated secondary goat anti-rabbit or anti-mouse IgG antibody (Leica). The slides were counterstained with 50% hematoxylin in water.
5
Honokiol Inhibits Tumor Growth in NOD/SCID Mice
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NOD/SCID mice were inoculated subcutaneously with 5 × 103 SAS SP cells into the flank and allowed to grow. Mice were randomly divided into four groups (n = 5): vehicle control (1 % carboxymethyl cellulose, CMC, Sigma) and honokiol-treated groups at different dose (20, 40, 80 mg/kg). Three weeks after inoculation, honokiol (diluted in 1 % CMC immediately prior to administration) was given intraperitoneally to mice thrice a week until week 10. At the end, mice were sacrificed and the tumors were paraffin embedded for the immunohistochemical staining of PCNA (PC10, mouse monoclonal antibody, #2586, Cell Signaling Technology, Beverly, USA) and CD31 (JC/70A, mouse monoclonal antibody, ab9498, Abcam, Cambridge, UK). The PCNA labeling index was calculated as the percentage of positively stained nuclei in a total of 600 cells in 3 different areas. The vascular density was determined by counting the number of CD31-positive microvessels per high-power field (x200) [32 (link)].
6
Immunostaining and Imaging of Tumouroids
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Tumouroids were fixed using 10% formalin for 30 minutes and washed with PBS. After permeabilising with 0.2% TX- 100 for 30 minutes, the gels were washed thoroughly with PBS (3 × 5 minutes) and blocked with 1% BSA for 30 minutes. After washing with PBS, gels were incubated with either a CK20 rabbit primary antibody (D9Z1Z from New England Biolabs, Herts, UK) or an anti-CD31 mouse primary antibody (JC70/A from Abcam, Cambridge, UK) overnight at 4 °C. After washing thoroughly, gels were incubated with an Alexa Fluor 488 goat anti mouse IgG secondary antibody (Invitrogen, Paisley, UK) and DyLight 594 goat anti-rabbit IgG secondary antibody (Vector labs, Peterborough, UK) for 2.5 hours at room temperature. The actin filaments were stained with Rhodamine Phalloidin conjugated to an Alexa Fluor® 488 fluorescent antibody (Invitrogen) for 1 hour at room temperature. The gels were then washed with PBS thoroughly and 2 drops of NucBlu (Invitrogen, Paisley, UK) was added to the PBS in each sample. Tumouroids were imaged using an inverted EVOS® FL imaging microscope (Invitrogen).
7
Histological Analysis of Tissue Samples
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The collected tissues were fixed in 4% paraformaldehyde (PFA, Servicebio, China), dehydrated with gradient alcohol series and embedded in paraffin. The embedded samples were cut into 6-µm-thick sections for hematoxylin and eosin (H&E) staining and Masson’s trichrome staining. Immunofluorescence (IF) staining was carried out as previously described [18 (link)]. The collected samples were fixed in 4% PFA, dehydrated with gradient series of sucrose and embedded in OCT. Ten-micrometer thick sections were incubated with primary antibodies, including those targeting AE1/AE3 (1:100; Thermo Fisher, USA, 53-9003-82), CD31 (1:100; Novus, Germany, JC/70A), and α-SMA (1:200; Abcam, UK, ab7817). Then, the sections were incubated with secondary antibodies at room temperature for 1 h. Fluorescence images were clearly observed under a Leica DM6B microscope.
8
Quantification of ICAM-1 and CD31 in ECs
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For the evaluation of the expression level of ICAM-1 and CD31 (PECAM-1) in ECs, mouse monoclonal anti-ICAM-1 and anti-CD31 antibodies (MEM-111 and JC/70A, Abcam, Cambridge, UK) were used with 1000× dilution. After the appropriate treatment, the EC monolayers were fixed with 10% formalin overnight at 4 °C and then permeabilized with 0.2% Triton X-100 for 10 min at room temperature. Following permeabilization, the cells were blocked with 10% goat serum for 2 h at room temperature and incubated with specific primary anti-ICAM-1 and anti-CD31 antibodies overnight at 4 °C. A Corresponding secondary antibody conjugated to Alexa 488 (Thermo Fisher Scientific, Rockford, USA; 1:1000) was applied for 1 h at room temperature. Cells were washed with PBS four times for 5 min, and the resulting fluorescence was quantified with a SpectraMax iD5 microplate reader (Molecular Devices, San Jose, USA). Hoechst 33342 dye was added to stain cell nuclei, and cell images were obtained using a Cytation 5 imaging plate reader (BioTek Instruments, Winooski, VT, USA) with specific objectives.
9
Immunohistochemical and Immunofluorescence Analyses of Tumor Angiogenesis
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Standard immunohistochemistry procedure [20 (link)] was followed with the following antibody dilutions: anti-Ki67 (Dako, clone MiB-1), 1:50; anti-CD31 (Dako, clone JC70A), 1:50; anti-CD105 (Abcam, ab49679), 1:100; anti-vWF, 1:800 (Abcam, ab6994). Standard immunofluorescence was followed with primary antibody dilutions of anti-CD31, anti-CD105 and anti-vWF as per immunohistochemistry; anti-GFAP (Abcam, ab726), 1:1000; anti-HIF-2α (Abcam, ab199) 1:100 and secondary antibody combinations of Alexa488-conjugated goat anti-rabbit, 1:200 and Alexa555-conjugated goat anti-mouse, 1:200. Images were taken using a Nikon ECLIPSE 90i light microscope fitted with a Hamamatsu OCRA-ER camera using three fluorescent light filters. For KNS42 cell cultures undergoing Hypoxyprobe™ (Hypoxyprobe Inc., Burlington, MA) analysis, the Hypoxyprobe™ reagent (pimonidazole) was applied two hours before harvesting at 200 μM concentration. Samples were then fixed as standard and immunohistochemistry performed using the primary antibody supplied with the Hypoxyprobe™ kit at 1:200 concentration. Three independent RCCS aggregates were fixed and sectioned prior to immunohistochemical detection and representative qualitative images taken from five slides.
10
Quantification of Muscle Microvascular Density
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