For TB infected lungs, histology mice were sacrificed by cervical dislocation, a slit was created in the trachea and one lung lobe placed in PBS for confirmation of CFU. A tube was inserted in the trachea and lungs were inflated with 1 ml of 10% Neutral Buffered Formalin (NBF; Sigma Aldrich, UK). The lungs were removed and placed in 10 ml of NBF overnight at 4 °C and then transferred into 70% ethanol and stored at 4 °C.
Paraffin-embedded tissue samples were sectioned (7 μm) and stained with hematoxylin and eosin (Sigma) and Acid Fast stain (BD Biosciences). Immunohistochemical staining was performed using anti-iNOS antibody (sc-651, Santa Cruz, 1/500) and anti-Galectin 3 (AF1197, R&D Systems, 1/500). This was followed by a secondary biotinylated antibody (Vector Laboratories, UK) then avidin–biotin–AP complex and visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories, UK).
Paraffin-embedded tissue samples were sectioned (7 μm) and stained with hematoxylin and eosin (Sigma) and Acid Fast stain (BD Biosciences). Immunohistochemical staining was performed using anti-iNOS antibody (sc-651, Santa Cruz, 1/500) and anti-Galectin 3 (AF1197, R&D Systems, 1/500). This was followed by a secondary biotinylated antibody (Vector Laboratories, UK) then avidin–biotin–AP complex and visualized with Vector Red alkaline phosphatase substrate (Vector Laboratories, UK).