Immunohistochemical Analysis of Tumor Angiogenesis

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%).

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Negrea G., Rauca V.F., Meszaros M.S., Patras L., Luput L., Licarete E., Toma V.A., Porfire A., Muntean D., Sesarman A, & Banciu M. (2022). Active Tumor-Targeting Nano-formulations Containing Simvastatin and Doxorubicin Inhibit Melanoma Growth and Angiogenesis. Frontiers in Pharmacology, 13, 870347.

Publication 2022

ab124432 MCA497 ABS82

Anti antibody Antibodies Antigens Cells Densitometry Differentiation marker Dilutions Macrophage Microscope Mouse Rabbit Stain Tumor microenvironment Vascular Vegf

Corresponding Organization : Technical University of Munich

Other organizations : National Institute of Research and Development for Biological Sciences, Iuliu Hațieganu University of Medicine and Pharmacy

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Variable analysis

independent variables

Nano-formulations

dependent variables

Effects on the development of new vasculature (measured by CD31 expression)
Invasive tumor cells in the tumor microenvironment (measured by VEGF expression)
Mature macrophages associated to the tumor microenvironment (measured by F4/80 expression)

control variables

Primary antibody dilutions: anti-CD31 (1:1,000), anti-F4/80 (1:250), and anti-VEGF (1:400)
Microscopy equipment: Optika trinocular microscope B383-FL with an MDC CCD Camera 2 MP
Image analysis: Adobe Photoshop CS6 and ImageJ software
Scoring system for immunohistochemical reaction: 0.5 (5–20%), 1 (20–40%), 2 (40–60%), 3 (60–80%), and 4 (80–100%)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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