Quantifying ZNFX1 and Autophagy Markers in TB

Paraffin-embedded TB patient lung and LN tissues were provided and sliced into 5 μm–thick sections by Guangzhou Chest Hospital with the written consent of all participants. Levels of ZNFX1 in vivo were detected with immunohistochemistry (IHC) staining according to the previously described protocol (57 (link)), with the antibody against ZNFX1 (1:50; catalog HPA046629; Merck Millipore) and HRP-conjugated goat anti-rabbit IgG (H+L) (1:200; catalog 31460; Thermo Fisher Scientific). Paraffin-embedded sections of murine lungs and spleens were stained with anti-F4/80 antibody (1:100; catalog MA5-16630; Thermo Fisher Scientific) together with anti-LC3 antibody (1:100; catalog 14600-1-AP; Proteintech) or anti–p-AMPK antibody (1:100; catalog AF3423; Affinity) at 4°C overnight, followed by staining with goat anti-mouse IgG 488 (catalog A28175) for F4/80 and goat anti-rabbit IgG 647 (catalog A27040) (Thermo Fisher Scientific) for LC3 and p-AMPK at room temperature for 1 hour. Samples were then scanned with CaseViewer2.4 (3DHISTECH). The levels of ZNFX1 in the TB tissues and the levels of LC3 puncta or p-AMPK in macrophages in murine lung and spleen tissues were determined using ImageJ 1.53 by a reader following a protocol blinded to the knockout versus WT experimental condition.