4.4. Immunohistochemical Analysis and Quantification of Microvessel Density
This protocol is extracted from research article:
Anti-Angiogenic Treatment in Pseudomyxoma Peritonei—Still a Strong Preclinical Rationale
Cancers (Basel), Jun 5, 2021; DOI: 10.3390/cancers13112819

Formalin-fixed paraffin-embedded sections from the main surgical specimens were used for immunohistochemical analysis. Immunostaining was performed using antibodies targeting CD31 (panendothelial marker) (1:40, #M0823, Dako, Glostrup, Denmark), CD105 (proliferating endothelium marker) (1:25, NCL-CD105, Novocastra/Leica, Newcastle upon Tyne, UK), HIF1α (hypoxia marker) (1:300, #H72320-150, BD Bioscience, San Jose, CA, USA), Ki67 (proliferation marker) (1:25, #M7240, Dako), FAP (protease expressed on activated myofibroblasts and some cancers) (1:100, #ab53066, Abcam) and SMA (smooth muscle and myofibroblast marker)(1:1500, #M0851, Dako). Visualization was achieved using the EnVision+ peroxidase system (Dako), as recently described [24]. Negative controls consisted of sections that underwent similar staining procedures with non-relevant rabbit immunoglobulins or a monoclonal antibody of the same isotype as the primary antibody used. For all antibodies, positive controls were included with satisfactory results. The immunoreactivity of Ki67 and HIF1α was quantified by scoring the staining from 0 to 5 according to the distribution of positive cells (0 = 0%, 1 = <1%, 2 = 1–10%, 3 = 11–33%, 4 = 34–66% and 5 = 67–100%). Immunostaining of CD105 and CD31 was used to determine MVD, by the so-called “hot-spot” method [25]. Vessel count was assessed by light microscopy in tumor areas containing the highest numbers of capillaries and was performed independently by two pathologists (WR and BD). Vessel count was performed at 400× magnification field and the average of three representative areas was determined as the MVD; minor disagreements with respect to scoring were solved by re-examination and establishment of consensus. For CD31 a vessel lumen was necessary for a structure to be defined as a vessel, whereas for CD105 this was not required.

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