Sab2701819

Antibody staining and TUNEL assay were performed as described previously (29 (link)). The following antibodies were used: antibodies against Dendra2 (1:1000; AB821, Evrogen, Moscow, Russia), phospho-4E-BP1 (Thr-37/46) (1:500; catalog no. 2855, Cell Signaling), Hnf4a (1:200; sc-6556, Santa Cruz Biotechnology, Inc.), Prox1 (1:500; ab5475, Chemicon), 2F11 (1:1000; ab71826, Abcam, Cambridge, MA), and PCNA (1:1000; SAB2701819, Sigma).

For detection of new vessel formation, the wound areas were analyzed using CD31 primary antibody (rabbit monoclonal primary antibody, SAB5500059, Sigma Aldrich, USA). For the detection of basal keratinocyte proliferation, the PCNA primary antibody was used (rabbit polyclonal Anti-Proliferating Cell Nuclear Antigen antibody, SAB2701819, Sigma-Aldrich, USA). For detection of adipose-derived MSCs (ADSCs) in cutaneous tissues after injection in both treated groups—group IV (DM+ADSCs) and group V (DM+ADSCs+PRP), the CD105 antibody (rabbit, polyclonal primary antibody, SAB1306487, Sigma Aldrich, USA) was used.
Briefly, after fixing, embedding in paraffin, and dewaxing, the tissue sections were blocked in 3% normal goat serum/0.3% Triton X-100/0.1% BSA (Sigma Aldrich) in PBS. The sections were then incubated for 24 h at 4°C with the primary antibody (primary) against CD31 & PCNA, respectively (1:100 dilution), followed by goat anti-mouse IgG (secondary) for each primary antibody for 1 h at room temperature. After hematoxylin staining, tissue sections were washed and then dehydrated with ethanol, treated with dimethylbenzene, and sealed for microscopic analysis by two blinded experienced investigators [34 ].

A liver sample from Ctenomys sp. 1 and cysticerci were fixed in 4% formalin and preserved in 70% ethanol; the liver was separated for histology and cysticerci for definitive preparations. A lung sample from Ctenomys sp. 2 was separated for histology. Liver samples from Ctenomys sp. 1 and C. haigi, and lung samples from Ctenomys sp. 2 were dehydrated, embedded in paraffin and sectioned at 3–5 μm following standard histological techniques. Sections were stained with haematoxylin–eosin (H&E), toluidine blue and Gomori's trichrome for morphological description. Other liver sections from Ctenomys sp. 1 were incubated separately with the primary antibodies pancytokeratin (Pk, dilution 1/250, Agilent, Z0622) to identify epithelial origin cells, and proliferating cell nuclear antigen (PCNA, 1/3000 dilution, Sigma-Aldrich, SAB2701819) to identify proliferating cells. All techniques used were based on standardized protocols of the Laboratorio de Histología y Embriología Descriptiva, Experimental y Comparada, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata (Acuña et al., 2021 (link)).