E3o7v

All animals were kept in our animal facility with free access to standard laboratory diet and water. No mice used for the longevity study were used for any other biochemical, physiological, or metabolic tests. The endpoint of life was the time when each mouse was found dead during daily inspection. Moribund mice were euthanized according to our institutional animal care guidelines, and the time at euthanasia was its endpoint. Survival data of each cohort were analyzed by plotting the Kaplan–Meier curve and performing the log-rank test using Prism. Tumor and organ tissues were dissected immediately after the animals were euthanized or dead, fixed with 10% formalin neutral buffer solution (Fujifilm) for 24 h, and processed for paraffin-embedded sections, followed by hematoxylin–eosin staining for pathological diagnosis. Immunostainings were performed, if necessary, for differentiation of tumors, by BOND MAX/III (Leica) with Bond Polymer Refine Detection (ds9800; Leica). Antibodies against CD68 (1:2,000 dilution; histiocytic marker; E3O7V; #97778; Cell Signaling Technology), alfa-fetoprotein (1:500 dilution; 14550-1-AP; Proteintech), CD45R (1:300 dilution; B-cell marker; B220; #550286; BD Biosciences; with F [ab’2] anti-rat IgG [H&L]; 1:500 dilution; 712-4126; Rockland antibodies & assays), and CD3 (1:500 dilution; T-cell marker; 21120-1-AP; Proteintech) were used.

Sixteen teeth from four dogs were extracted. Premolars were divided into mesial and distal roots to obtain two samples from one tooth. The extracted tooth sample was fixed in 4% paraformaldehyde (PFA) at 4°C for 24 h, decalcified with Kalkitox™ at 4°C for 48 h, neutralized in 5% sodium sulfate solution for 24 h (all purchased from FUJIFILM Wako Pure Chemical), and embedded in paraffin. Standard hematoxylin and eosin (H&E) staining was performed as previously described [16 (link)]. For immunohistochemical staining, the specimens were probed with specific primary antibodies against myeloperoxidase (MPO) (1 : 1000, ab208670; Abcam), CD68 (1 : 150, E3O7V; Cell Signaling Technology), and pPKR (phospho T446) (1 : 200, ab32036; Abcam) and counterstained with Mayer's hematoxylin solution (FUJIFILM Wako Pure Chemical).