Retrievagen a

Immunostaining was performed on the liver sections following antigen retrieval using Retrievagen A (Zymed Laboratories, Inc., San Francisco, CA, USA) at 100°C for 20 min, and endogenous peroxidases were quenched with 3% H₂O₂ (Tianjin Jinqiang Chemical Co., Ltd.). The sections were blocked with 2% bovine serum albumin (BSA) in phosphate-buffered salin (PBS) followed by staining with primary anti-cleaved caspase-3 (BD Pharmingen, San Jose, CA, USA) at room temperature for 1 h. The sections were washed and following the application of the secondary antibody (R&D Systems, Inc.), the tissues were developed using Vectastain ABC and 3,3′-diaminobenzidine (Vector Laboratories, Inc., Burlingame, CA, USA). Following staining, five high-power fields (magnification, ×200) were randomly selected in each slide, and the average proportion of positive expression in each field was counted using the true color multi-functional cell image analysis management system (Image-Pro Plus; Media Cybernetics, Inc., Rockville, MD, USA), and expressed as positive unit (pu).

Immunostaining was performed on the lung sections after Ag retrieval by using the Retrievagen A (Zymed, South San Francisco, CA) at 100°C for 20 min, and endogenous peroxidases were quenched with 3% H₂O₂. Sections were blocked with 2% BSA in PBS and stained using primary anti-NF-κB65 and TLR4 (Bioworld Technology, Inc., St. Louis Park, MN, USA) at room temperature for 1 h. then sections were washed and applied with the secondary Ab (R&D Systems Minneapolis, MN, USA). The tissues were developed using the Vectastain ABC (Vector Labs, Burlingame, CA) and 3,3′-diaminobenzidine (Vector Labs). NF-κB65- and TLR4-positive expression in the lung tissue was determined and expressed as positive units by using the Image-Pro Plus image analysis software (Media Cy Bemetics American Company).

Lungs were inflation fixed at a constant pressure (25 cm H₂O) by tracheal installation of 4% paraformaldehyde, transferred to 70% ethanol after 24 h and embedded in paraffin as previously described (19 (link)). Immunostaining was performed on lung sections after antigen retrieval using Retrievagen A (Zymed, San Francisco, CA, USA) at 100°C for 20 min, and quenching endogenous peroxidases with 3% H₂O₂. Sections were blocked with 2% bovine serum albumin in PBS, followed by staining with primary anti-α-SMA (1:2,000; cat. no. 11475; BD Pharmingen, San Jose, CA, USA) at room temperature for 1 h. Sections were washed and after application of the goat HRP-conjugated anti-rabbit IgG secondary antibody (1:4,000; cat. no. HAF109; R&D Systems), tissues were developed using Vectastain ABC (Vector Labs, Burlingame, CA, USA) and 3,3′-diaminobenzidine (Vector Labs). After staining, five high-power fields (magnification, ×400) were randomly selected in each slide, and the average proportion of cells with positive expression in each field was counted using the true color multi-functional cell image analysis management system (Beckman Coulter, Inc, Fullerton, CA, USA), with values expressed as positive units (pu).