Citrisolve

As previously described, representative liver, kidney, heart, brain, stomach, spleen, lung, uterus, urinary bladder, cecum, and small and large intestine samples were collected during necropsy and fixed in 10% buffered formalin for 24 h [18 (link)]. The tissues were subsequently transferred to 70% ethanol prior to being processed and embedded. Sections were stained with hematoxylin and eosin for histopathological evaluation. For immunohistochemistry (IHC), 3–4 μm sections were deparaffinized in CitriSolve (Decon Labs., King of Prussia, PA, USA) and rehydrated by processing through graded alcohols. Tissues were pretreated with Proteinase K (10 μg/mL) for 15 min. Endogenous IgG and non-specific background were blocked with Rodent Block M (Biocare Medical; Pacheco, CA, USA) for 20 min, followed by an alkaline phosphatase block (BLOXALL; Vector Laboratories, Burlingame, CA, USA) for 10 min. The primary antibody (PA1-7206; Thermo Fisher Scientific, Waltham, MA, USA) was incubated on the tissue sections at a dilution of 1:10,000 for 1 h at room temperature. Sections were sequentially incubated with a rabbit-on-rodent tissue alkaline phosphatase-based polymer and Warp Red (Biocare Medical, Pacheco, CA, USA). Tissues were counterstained with Tacha’s hematoxylin and mounted using EcoMount (Biocare Medical, Pacheco, CA, USA) [18 (link)].

Tissue sections, 3–4 μm in thickness, were deparaffinized in CitriSolve (Decon Labs., King of Prussia, PA) and rehydrated by processing them through graded alcohol solutions. Antigen unmasking was accomplished by digesting tissue in 10 μg/ml of protein kinase for 15 min at room temperature (Fisher Scientific, Waltham, MA). Endogenous enzymes and non-specific background were blocked with Background Punisher (Biocare Medical, Pacheco, CA), followed by BLOXALL (Vector Laboratories, Burlingame, CA). The primary antibody (NBP1–42140; Novus Biologicals, Centennial, CO) was incubated on the tissue sections at a dilution of 1:100 for 1 h at room temperature. Subsequently, sections were sequentially incubated with an alkaline phosphatase-based detection polymer kit (MACH 4; Biocare Medical), and Warp Red (Biocare Medical). Sections were counterstained with Tacha’s hematoxylin (Biocare Medical) and mounted using a permanent mounting medium (EcoMount; Biocare Medical).