Formalin-fixed paraffin-embedded (FFPE) sections from archival xenografts established with shNT or shERK5–2 A375 or SSM2c cells, or with A375 cells from XMD8–92 (25 mg/kg)- or vehicle (2-hidroxypropyl-β-cyclodextrin 30%)-treated mice were used (15 (link)). Experiments had been approved by the Italian Ministry of Health (authorization no. 213/2015-PR) and were in accordance with the Italian ethic guidelines and regulations. Sections (3 μmol/L thick) were deparaffinized and stained with Sudan Black Blue (SBB; Bio-Optica) to reveal lipofuscin (28, 29 (link)), and counterstained with Nuclear Fast Red (NFR, Bio-Optica). Immunohistochemistry (IHC): After citrate buffer antigen retrieval, staining was performed with the UltraVision LP Detection System HRP Polymer Kit (Thermo Fisher Scientific) following the manufacturer's instructions. Sections were incubated overnight at 4°C with primary antibodies (Supplementary Table S2) and (3,3′-diaminobenzidine; Thermo Fisher Scientific; DAB) used as a chromogen. Sections were counterstained with hematoxylin.
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Immunohistochemical Analysis of FFPE Xenografts
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Antibodies
Antigen
Buffer
Cells
Chromogen
Citrate
Cyclodextrin
Embedded paraffin
Formalin
Hematoxylin
Immunohistochemistry
Lipofuscin
Mice
Polymer
Xenografts
Xmd8 92
Corresponding Organization : University of Florence
Other organizations : Piedmont Reference Center for Epidemiology and Cancer Prevention, Centro de Investigación del Cáncer, Instituto de Investigación Biomédica de Salamanca, Centro de Investigación Biomédica en Red de Cáncer
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Variable analysis
independent variables
- Knockdown of ERK5 (shERK5–2)
- Treatment with XMD8–92 (25 mg/kg)
- Lipofuscin levels (revealed by Sudan Black Blue staining)
- Unspecified immunohistochemistry (IHC) outcomes
- Formalin-fixed paraffin-embedded (FFPE) sections from archival xenografts
- Sections (3 μmol/L thick)
- Citrate buffer antigen retrieval
- Staining with Nuclear Fast Red (NFR)
- Counterstaining with hematoxylin
- Negative control: shNT (non-targeting shRNA)
- Negative control: Vehicle (2-hidroxypropyl-β-cyclodextrin 30%)
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