2.7.5. Immunohistochemistry

Immunohistochemical studies were performed on para ffin-embedded explant tissue sections of 5 nm thickness, previously depara ffinized and rehydrated using a standard technique. Rabbit polyclonal anti-tumor necrosis factor (TNF)- α diluted 1:100 (Santa Cruz, California) was used as a primary antibody.

Immunoreactions were visualized employing a Novocastra Peroxidase/DAB kit (Leica Biosystems, Nussloch, Germany), according to the manufacturer's instructions. Negative control sections were processed by the substitution of primary antibodies with irrelevant immunoglobulins of matched isotype used in the same conditions as primary antibodies. Stained slides were analyzed under bright-field microscopy.
