Sp 9001 splink detection kits

Kidney tissues were fixed in 10% buffered formalin solution for 30 min at room temperature and dehydrated in 75% ethanol overnight, followed by paraffin embedding. Immunohistochemical analysis was performed using the SP-9001 SPlink Detection kits (OriGene Technologies, Inc.), according to the manufacturers' instructions. Paraffin-embedded sections (4 µm) were deparaffinized with xylene and subsequently rehydrated in a descending series of ethanol washes (100, 90, 85 and 75%; 5 min each). The sections were treated for 15 min at 98°C with 3% H₂O₂ in methanol to inactivate the endogenous peroxidase activity and were subsequently incubated at room temperature for 1 h with primary antibodies rabbit against collagen I (cat. no. 14695-1-AP; 1:200; ProteinTech Group, Inc.), collagen III (cat. no. 22734-1-AP; 1:200; ProteinTech Group, Inc.), LC3 (cat. no. 14600-1-AP; 1:200; ProteinTech Group, Inc.) and p62 (cat. no. 55274-1-AP; 1:200; ProteinTech Group, Inc.). The secondary antibody from SPlink Detection kits (OriGene Technologies, Inc.) was incubated with the tissue for 30 min at room temperature. All sections were examined using a BX40 upright light microscope (Olympus BX43; Olympus Corporation). For each staining, 3×7 sections (6 mice) per group were analyzed and the representative images were presented. All image analyses were performed by a blinded reviewer.