Biotinylated secondary goat anti rabbit igg

The slides were deparaffinized and rehydrated before staining. To avoid nonspecific antibody binding, the slices were incubated in blocking solution (Vector Laboratories) for 1 h and reacted with anti-rat syndecan-1 antibody (Santa Cruz Biotechnology, Dallas, TX, USA) at a 1:100 dilution overnight at 4 °C. The slides were washed in PBS, and the sections were incubated for 1 h with biotinylated secondary goat anti-rabbit IgG (Abcam, Cambridge, UK). After incubation, ABC Reagent (Vector Laboratories) was applied to react with the biotinylated antibody for 1 h at 25 °C and attached with the 3,3′-diaminobenzidine (DAB) reagent (Vector Laboratories). Mean staining intensity without DAB for syndecan-1 was standardized as a negative control. The slides were stained with haematoxylin as a counterstain, dehydrated, and cover-slipped using mounting medium (Vector Laboratories). Images were obtained through a microscope. Syndecan-1 intensity was quantified using ImageJ software.

Toll-like receptor-4 (TLR4) is a transmembrane protein, and its activation leads to an intracellular signaling pathway and production of inflammatory cytokines, responsible for activating the innate immune system. Therefore, TLR4 was used to check the immunity in the tissues. The slides were deparaffinized and rehydrated. To avoid non-specific binding of antibody, the slices were incubated in blocking solution (Vector Laboratories, Burlingame, CA, USA) for 1 h and reacted with TLR4 rabbit polyclonal antibody (Abcam, Cambridge, UK) at a 1:100 dilution overnight at 4°C. The slides were then washed in PBS, and the sections were incubated for 1 h with biotinylated secondary goat anti-rabbit IgG (Abcam). After the incubation, the ABC Reagent (Vector Laboratories) was applied to react with the biotinylated antibody for 1 h at 25°C and attached with 3,3`-diaminobenzidine reagent (Vector Laboratories). The slides were stained with hematoxylin as a counterstain, dehydrated and cover-slipped using mounting medium (Vector Laboratories). Images were obtained under a microscope (Nikon, Tokyo, Japan). TLR4 intensity was quantified using Image J software (NIH, Bethesda, MD, USA).