Tnf α

Cytokines in the BAL and serum were quantified with enzyme-linked immunosorbent assays (ELISA) following the manufacturer’s instructions of the following kits for the guinea pigs: TNF-α (MBS9303082; MyBioSource, San Diego, CA, USA), IFN-γ (MBS701377; MyBioSource, San Diego, CA, USA), TGF-β1 (CSB-E06773GU; CUSABIO TECHNOLOGY LLC; Houston, TX, USA), IL-1β (MBS765173; MyBioSource, San Diego, CA, USA), 1L-6 (MBS269054; MyBioSource, San Diego, CA, USA) IL-8 (MBS282965; MyBioSource, San Diego, CA, USA), IL-12 (MBS704591; MyBioSource, San Diego, CA, USA).

IHC analysis was conducted as previously described [28]. Deparaffinized tissue sections were treated with 3% hydrogen peroxide in methanol for 10 min to remove endogenous peroxidase. Antigen retrieval was carried out with sodium citrate buffer (0.1 M) using the microwave method. The slides were incubated with normal serum to block nonspecific binding and then incubated for 1 h with primary antibodies (diluted 1:100 to 1:200) such as IFN-γ (sc-74104, Santa Cruz Biotechnology, Dallas, TX, USA), IL-12p40 (sc-57258), IL-4 (sc-73318), IL-13 (sc-1776), TNF-α (MyBioSource, San Diego, CA, USA), and IL-6 (Novus Biologicals, Littleton, CO, USA). The slides were incubated for 10 min with biotinylated secondary antibodies (Vector Laboratories, PK-7800, Burlingame, CA, USA) and horseradish peroxidase-conjugated streptavidin. Signals were detected using the 3,3-diaminobenzidine tetrahydrochloride substrate chromogen solution, and the cells were counterstained with Mayer’s hematoxylin.

Commercially available ELISA kits designed for samples of chicken origin were purchased from MyBioSource (San Diego, CA, USA) for the quantification of selected pro-inflammatory factors including tumor necrosis factor alpha (TNF-α; Cat. # MBS746318), interleukin-1 beta (IL-1β; Cat. # MBS454453), interleukin-6 (IL-6; Cat. # MBS2021018) and C-reactive protein (CRP; Cat. # MBS764341), as well as selected proteins with antibacterial properties comprising cathelicidin (Cat. # MBS735193), beta-defensin (Cat. # MBS018020) and lysozyme (Cat. # MBS701562). A double-sandwich ELISA protocol was performed according to the instructions of the manufacturer and the absorbances were read with the help of the GloMax plate spectrophotometer (Promega, Madison, WI, USA) at 450 nm.