The immunohistochemical analysis was performed as previously described (Luput et al., 2018 (link)) for the following antigens: CD31, a marker for vascular differentiation, to show the effects of the nano-formulations on the development of new vasculature; VEGF, to stain the invasive tumor cells in the tumor microenvironment (TME); and F4/80, to highlight the mature macrophages associated to the TME. The following dilutions of the primary antibodies were used: anti-CD31 antibody (rabbit IgG anti-mouse CD31, Abcam, ab124432) diluted 1:1,000, anti-F4/80 mouse macrophage receptor (rat IgG anti-mouse F4/80, Bio-Rad, MCA497) diluted 1:250, and anti-VEGF (rabbit IgG polyclonal/anti-mouse VEGF, Sigma-Aldrich, ABS82) diluted 1:400. Then, the slides were blindly investigated by histologists using an Optika trinocular microscope B383-FL with an MDC CCD Camera 2 MP. After microphotography, the images were prepared in Adobe Photoshop CS6 software in order to generate a whole figure. The area of positive immunoreaction was determined by color densitometry plug-in using Image J software. The scoring system used for immunohistochemical reaction was 0.5 (5–20%); 1 (20–40%); 2 (40–60%); 3 (60–80%); and 4 (80–100%).
Ab124432
Manufactured by Abcam
Sourced in United Kingdom, United States
Ab124432 is a laboratory equipment product offered by Abcam. The core function of this product is to [PRODUCT_DESCRIPTION].
Automatically generated - may contain errors
Lab products found in correlation
Mca497, by Bio-Rad (3 mentions)
Ab16667, by Abcam (3 mentions)
Ab7817, by Abcam (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Abs82, by Merck Group (1 mentions)
395b 1kt, by Merck Group (1 mentions)
Ds ri2, by Nikon (1 mentions)
Ab16694, by Abcam (1 mentions)
Ab108595, by Abcam (1 mentions)
Anti β tubulin antibody, by Santa Cruz Biotechnology (1 mentions)
Anti e cadherin antibody, by BD (1 mentions)
Sb 505124, by Merck Group (1 mentions)
Sb431542, by Merck Group (1 mentions)
Ldn193189, by Merck Group (1 mentions)
Bmp 2, by R&D Systems (1 mentions)
Rotary microtome, by Leica (1 mentions)
Ab53444, by Abcam (1 mentions)
Ab210826, by Abcam (1 mentions)
Ab125212, by Abcam (1 mentions)
29 protocols using ab124432
1
Immunohistochemical Analysis of Tumor Angiogenesis
2
Dual Immunohistochemistry for PAS and CD31
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PAS and CD31 detection in FFPE slides was conducted as previously described [54 (link),55 (link)]. The slides were first immunostained with CD31 (Abcam, Cambridge, UK, ab124432) as described above. After secondary antibody incubation, PAS staining was performed following the manufacturer’s recommendations (Sigma, St. Louis, MO, USA, 395B-1KT). Briefly, the slides were incubated for 5 min at room temperature with PAS, washed, and incubated for 15 min with Schiff’s Reagent. The slides were then counterstained with hematoxylin, dehydrated, and mounted.
3
Immunohistochemical Analysis of Cleared Tissues
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The optically cleared tissue samples were immersed in anhydrous methanol (2 × 99.8% (vol/vol) for 3 h at RT) to remove the BABB clearing solution after LSFM acquisition. Specimen were then processed to paraffin-embedded tissue blocks and immunohistochemistry (α-PECAM-1 (diluted 1:1000), ab124432, Abcam plc., Cambridge, UK) was performed on microtome sections. Paraffin sections were imaged with a Nikon Ti2-E epifluorescence microscope (Nikon BV, Amsterdam, NL), 20 × Plan Fluor objective (NA = 0.45) and a color camera (Nikon DS-Ri2). For image analysis Nikon NIS–Elements and FIJI by ImageJ53 (link) were used.
4
Inhibiting Tumor Growth Signaling Pathways
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Anti-gremlin-1 antibody was from Origene (TA324077), anti-E-cadherin antibody from BD Biosciences (610182) and anti-β-tubulin antibody from Santa Cruz (sc-9104). Integrin alpha v blocking antibody was from Millipore (MABT207) and used at 10 μg/ml concentration. Antibodies used for immunohistochemistry were anti-human calretinin (Abcam, ab16694), anti-mouse CD31 (Abcam, ab124432), anti-Ki67 (Abcam, ab16667) and anti-human laminA+C nuclear envelope marker (Abcam, ab108595), which was used to identify human tumor cells in mouse tissue. Broad spectrum MMP inhibitors GM6001 and BB2516 were from Calbiochem and used at 10 μM concentration. TGF-βR inhibitors SB431542 and SB505124 were from Sigma-Aldrich and used at 10 μM concentration. BMP receptor inhibitor LDN193189 was from Sigma-Aldrich and used at 100 nM concentration. BMP-2 was from R&D Systems and used at 25 ng/ml concentration. The recombinant fusion protein containing the ectodomain of human ActR2B (sActR2B-Fc) or anti-Müllerian hormone receptor (sAMHR2-Fc) fused to the Fc domain of human IgG1 and recombinant follistatin were produced as described previously [40 (link)] and used at 10 μg/ml. Soluble receptors sequester ligands and inhibit their binding to cell surface receptors.
5
Histological Analysis of Tumor Microenvironment
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For histological analysis, tumors were paraffin-embedded and sectioned in 5 µm tissue slices using a rotary microtome (Leica Microsystems, Nussloch, Germany). Hematoxylin and eosin (H&E) staining was performed according to standard protocols and enabled identification of intratumoral hemorrhage and necrosis. Immunohistochemical staining of CD31, a marker for vessel density and angiogenesis [40 (link)], was performed as previously described [41 (link)]. In brief, it was started with dewaxing and rehydration, followed by incubation in 3% H2O2 for 10 min. Afterward, sections were incubated with primary antibody CD31 (ab124432, Abcam, Cambridge, UK) for one hour at 4 °C in a dilution of 1:200 in blocking buffer, followed by horseradish peroxidase/diaminobenzidine detection. To analyze changes of the intratumoral immune cell infiltrate after immune checkpoint blockade, further immunohistochemical staining of CD3 (T-cells), F4/80 (macrophages), Ly6G (neutrophils), CD49b (natural killer cells) and CD45R (B-cells) was performed (Additional file 1 : Meth. 1). For each group, n = 3 samples have been analyzed qualitatively.
6
Aortic Tissue Immunostaining Analysis
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The harvested aortic roots were embedded and sliced. Quantitative immunostaining was performed for macrophages, vascular endothelial cells, and smooth muscle cells in frozen slides of the aorta using an anti-CD68 antibody (ab53444, Abcam, USA), anti-α-SMA antibody (ab7817, Abcam, USA), anti-TXNIP antibody (ab210826, Abcam, USA), and anti-CD31 antibody (ab124432, Abcam, USA). Macrophage, smooth muscle cell, vascular endothelial cell, and TXNIP staining was expressed as the fluorescence intensity of the CD68-, α-SMA-, CD31-, and TXNIP-positive areas respectively.
7
Immunohistochemical Staining Protocol
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For IHC staining, the hydrated sections were treated with citrate antigen retrieval solution (Beyotime, Shanghai, China) at 95 °C for 15min and cooled down at RT. 5% goat serum in PBS was used to block the nonspecific binding sites after inactivating endogenous peroxidase. Primary antibodies of anti-Ki67 (ab16667, 1:400), or anti-CD68 (ab125212, 1:2000), or anti-CD31 (ab124432, 1:1000) from Abcam were used for overnight staining at 4 °C. Next, sections were incubated with secondary antibody of goat anti-rabbit IgG (Zhongshan Golgen Bridge Biotechnology, Beijing, China) for 30 min at RT. Finally, the sections were stained with fresh diaminobenzidine (Zhongshan Golgen Bridge Biotechnology) and counterstained with hematoxylin. All images were taken using the ScanScope slide scanner with Aperio ImageScope analysis software.
8
Kidney Protein Expression Analysis
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Kidneys were isolated and homogenized on ice using a glass douceur in RIPA buffer (Thermo, 89900). Protein concentration was quantified using the Pierce BCA Protein Assay Kit (Thermo, 23225) following the manufacturer’s recommendations. Equal amounts of protein were resolved using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, and detected by Western blot using rabbit anti-CD31 polyclonal antibodies (Abcam, ab124432) or anti-β-actin mAb (Abcam, ab8226). After washing, bound primary antibodies were detected by horseradish peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence (ECL) Plus Western Blotting Substrate (Thermo, 32134).
9
Immunofluorescence Staining for CD31 and Na+/K+-ATPase
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Antibodies against anti-CD31 (ab124432) and Na+/K+-ATPase α1 (ab7671) were purchased from (Abcam, Cambridge, UK); cleaved caspase-3 (9961) was purchased from (Cell Signaling Technology, MA, USA).
10
Analyzing Vein Graft Lesions and Neoangiogenesis
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After euthanasia, vein graft segments were collected, fixed in 4% paraformaldehyde (PFA) for 24 h, dehydrated overnight in 60% isopropanol, and subsequently embedded in paraffin. Cross sections of vein graft segments were stained with hematoxylin and eosin to evaluate lumen and lesion area, plaque thickness, and percentage of vein stenosis. Neovessels were detected inside vein graft lesions via standard immunohistochemistry using anti-CD31 antibody (endothelial cells; ab124432, Abcam). Anti-TER-119 (550,565, BD Biosciences) was used to determine plaque hemorrhages and anti-α-smooth muscle actin (α-SMA) (A2547, Sigma-Aldrich) was used to determine vascular smooth muscle cell (VSMC) coverage of neovessels. Anti-MAC3 (550,292, Pharmingen), a Masson’s Trichrome stain and anti-vascular cell adhesion molecule-1 (VCAM-1) (ab134047, Abcam) were used to stain macrophages, collagen, and VCAM-1 positive ECs, respectively.
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