*4.6. Histology and Immunohistochemistry Analysis*

Animal hind paws were collected, fixed in formalin 10% for 48h, and then decalcified in 10% EDTA solution, for 1 month. Tissue was embedded in paraffin and sections of 4 μm thickness were cut and stained with hematoxylin and eosin (H&E). Spleen and draining lymph nodes were fixed for 24 h, paraffin embedded and 3 μm thickness paraffin sections were cut for H&E staining and immunohistochemistry (IHQ).

Expression of proliferation marker Ki-67 and macrophage marker CD68 was assessed by IHQ. NovolinkTM Polymer Detection Kit (Leica Biosystems, Newcastle, UK) was used following the manufacturer's instructions. Antigen retrieval was performed by sections incubation in citrate buffer (98 ◦C, citrate buffer 10 mM, pH 6.0). After endogenous peroxidase neutralization, permeabilization with Triton X-100 0.3% was performed for Ki-67 immunostaining. Sections were incubated with Block protein solution and then in primary antibodies: anti-Ki-67 (1:50, clone SP6, ThermoFisher Scientific, Waltham, MA, USA) and anti-CD68 (1:100, clone ED1, Bio-Rad Laboratories, Irvine, CA, USA) for 45 min at RT. Positive staining was revealed after 30 min of incubation with NovoLinkTM Polymer and 5 min of incubation with peroxidase-substrate solution DAB. Finally, sections were counterstained with hematoxylin and slides mounted. Sections incubated without primary antibody were used as negative controls. Representative images of positive staining were taken using Olympus CX31 light microscope (20× objective). Nine images per slide were analyzed using Fiji software, and the number of nuclei Ki-67<sup>+</sup> and Ki-67<sup>−</sup> nuclei were counted. Proliferation index was calculated as the ratio of Ki-67<sup>+</sup> nuclei to the total number of nuclei counted.
