2.9. Histology, Immunohistochemistry and Morphometry

For histological analysis, the tumor, lung, liver and kidney specimens were collected and fixed in 10% neutral-buffered formalin, dehydrated in ascending ethanols and xylols and embedded in HISTOMIX paraffin (BioVitrum, St. Petersbur, Russia). Paraffin sections (5 μm) were sliced using a Microm HM 355S microtome (Thermo Fisher Scientific, Waltham, MA, USA) and stained with hematoxylin and eosin. For immunohistochemical (IHC) study, tumor sections (3–4 μm) were deparaffinized and rehydrated; antigen retrieval was carried out after exposure in a microwave oven at 700 W. The samples were incubated with anti-PCNA-specific antibodies (ab92552, Abcam, Cambridge, UK) according to the manufacturer’s protocol. Next, the sections were incubated with secondary horseradish peroxidase (HPR)-conjugated antibodies, exposed to the 3,3′-diaminobenzidine (DAB) substrate (Rabbit Specific HRP/DAB (ABC) Detection IHC Kit, ab64261, Abcam, Cambridge, UK), and stained with Mayer’s hematoxylin. All the images were examined and scanned using Axiostar Plus microscope equipped with Axiocam MRc5 digital camera (Zeiss, Germany) at magnification ×400.

Inhibition of metastases development of B16 melanoma in lungs of mice was assessed by morphometric analysis using the metastasis inhibition index (MII), calculated as MII = ((mean metastasis area control–mean metastasis area experiment)/mean metastasis area control) × 100%. The MII of the µ-Scr-treated group was taken as 0% and the MII that reflected the absence of metastases was taken as 100%.

Morphometric analysis of tumor and liver sections was performed using a counting grid consisting of 100 testing points in a testing area equal to 3.2 × 10⁶ μm². A total of 5–10 random fields were studied in each sample depending on sample size, forming 50–100 random fields for each group of mice in total. Morphometric analysis of tumor tissue included evaluation of the numerical density (Nv) of mitoses and the number of PCNA-positive cells with subsequent calculation of proliferation index according to the formula: number of PCNA-positive cells/total number of cells × 100%. Morphometric analysis of liver tissue included evaluation of the volume densities (Vv, %) of normal hepatocytes, dystrophy and necrosis in the liver parenchyma, as well as the numerical densities (Nv) of binuclear hepatocytes reflecting the regeneration capacity of the liver. The volume density (Vv, %) of studied histological structures representing the volume fraction of tissue occupied by this compartment was calculated using the formula: Vv = (Pstructure/Ptest) × 100%, where Pstructure denotes the number of testing points over the structure and Ptest denotes the total number of testing points—in this case, 100. The numerical density (Nv) of studied histological structures indicating the number of particles in the volume unit of the tissue was evaluated as a number of particles in the square unit, 3.2 × 10⁶ μm² in this case.