IHC staining was performed on formalin-fixed, paraffin-embedded tissue microarrays of 37 osteosarcoma patients’ samples (25 (link), 26 (link)), 19 PDX models, and 14 normal human tissue samples. Sections were stained with mouse or rabbit anti-human monoclonal antibodies against MT1-MMP (Abcam, ab51074, 1:400), MRC2 (Abcam, ab70132, 1:200), and CD276 (Abcam, ab219648, 1:100). All sections were counterstained with hematoxylin, dehydrated, and mounted. Sections were processed with peroxidase-conjugated avidin/biotin and 3,3’-diaminobenzidine (DAB) substrate (Leica Microsystems). Slides were scanned and digitalized using a ScanScope XT system (Aperio/Leica Technologies). The quantitative analysis of the IHC staining was performed by a pathologist (RL) using microscope direct observation. The results were expressed as membrane and cytoplasmic positivity in tumor cells, PDX, and normal tissue in the form of the H-score (intensity × % cells; 0 to 300). Cytoplasm staining was also observed. Representative images were obtained from scanned IHC slides with Halo software v3.1.1 (Indica Labs, RRID:SCR_018350).
Ab219648
Manufactured by Abcam
Sourced in United States
Ab219648 is a primary antibody that recognizes the Cytokeratin 18 protein. Cytokeratin 18 is a member of the keratin family of intermediate filament proteins and is commonly used as a marker for epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Halo software v3, by Indica Labs (2 mentions)
Ab51074, by Abcam (2 mentions)
Ab70132, by Abcam (2 mentions)
Peroxidase conjugated avidin biotin and 3 3 diaminobenzidine dab substrate, by Leica (1 mentions)
Ab228462, by Abcam (1 mentions)
Ab213363, by Abcam (1 mentions)
Ab218810, by Abcam (1 mentions)
Ab40763, by Abcam (1 mentions)
4 protocols using ab219648
1
Immunohistochemical Analysis of Osteosarcoma
2
CD276 Expression in Glioma Pathology
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Immunohistochemistry analysis (IHC) assays were performed to assess the expression of CD276 using a Rabbit monoclonal [EPR20115] antibody against CD276 (1:80,000, Abcam, ab219648) on 144 cases of glioma pathological sections. The assays were conducted using a biotin assay system (Beijing Zhongshan Jinqiao, China, Cat. No. PV-9001, PV-9002), following the manufacturer's instructions. For IHC analysis, the grading system utilized the following criteria: absence of staining denoted a score of 0, yellow staining denoted a score of 1, and brown staining denoted a score of 2. Based on the percentage of positively stained tumor cells in the visual field, a score of 0 was assigned for < 1% cells, a score of 1 for 1–25%, a score of 2 for 25–75%, and a score of 3 for 75–100%. The overall score was determined as the product of the intensity score and the percentage of positive cells.
3
IHC Analysis of MT1-MMP, MRC2, and CD276 in Osteosarcoma
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IHC staining was performed on formalin-fixed, paraffin-embedded tissue microarrays of samples of 37 patients with osteosarcoma (25, 26 (link)), 19 PDX models, and 14 normal human tissue samples. Sections were stained with mouse or rabbit anti-human mAbs against MT1-MMP (Abcam, ab51074, 1:400), MRC2 (Abcam, ab70132, 1:200), and CD276 (Abcam, ab219648, 1:100). All sections were counterstained with hematoxylin, dehydrated, and mounted. Sections were processed with peroxidase-conjugated avidin/biotin and 3,3′-diaminobenzidine substrate (Leica Microsystems). Slides were scanned and digitalized using a ScanScope XT system (Aperio/Leica Technologies). The quantitative analysis of the IHC staining was performed by a pathologist (R. Lazcano) using microscope direct observation. The results were expressed as membrane and cytoplasmic positivity in tumor cells, PDX, and normal tissue in the form of the H-score (intensity × % cells; 0 to 300). Cytoplasm staining was also observed. Representative images were obtained from scanned IHC slides with Halo software v3.1.1 (Indica Labs, RRID:SCR_018350).
4
Immunohistochemical Profiling of Immune Checkpoint Molecules
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The DGs tissues were fixed, dehydrated, and paraffin-embedded. The 4 μm sections were deparaffinized and rehydrated in 100, 95, and 75% ethanol. The antigen retrieval solution with EDTA (pH 9.0) was used for PD-L1 staining, while the solution with sodium citrate (pH 6.0) for B7-H3 and CD47 staining. After endogenous peroxidase activity blocking, the sections were rinsed and incubated with the primary antibodies including anti-human PD-L1 (1:1,000, Abcam, ab228462, Cambridge, MA, USA), CD47 (1:2000, Abcam, ab218810) and B7-H3 (1:2,000, Abcam, ab219648) overnight at 4°C. TILs were stained by the CD45 (1:1,000, Abcam, ab40763) antibody, and TAMs were stained by the CD68 antibody (1:1,000, Abcam, ab213363). Images were acquired after the incubation with secondary antibodies and DAB, and counterstained with Hematoxylin. The expression level of three immune checkpoint molecules was determined by the percentage of positive cells and the staining intensity: low (negative intensity and intensity 1, and intensity 2 with positive cells < 10%) and high (intensity 2 with positive cells ≥ 10% and intensity 3) expression (19 (link)). The expression of TILs/TAMs was similarly evaluated as previously described (20 (link), 21 (link)).
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