3 3 diaminobenzidine (dab)

Lung tissues from Mtb-infected mice were fixed in 4% phosphate-buffered formalin for 24 h and were then embedded in paraffin wax. The paraffin-embedded lungs were cut in serial sections with a thickness of 2–3 μm. Hematoxylin and eosin (H&E) staining were applied to detect the infiltration in the lungs. Acid-fast staining with the standard Ziehl–Neelsen method was employed for the determination of Mtb burden in the lungs. Five noncontiguous sections were examined for each lung, and at least five animals were sacrificed for each group. The stained slides were visualized by light microscopy.
For immunohistochemistry, lung tissue from three PTB patients with pulmonary lobectomy and six Mtb-infected mice was investigated. Segments of lung tissues were fixed in 10% buffered formalin and were embedded in paraffin. The blocks were cut into 5 μm sections, and five noncontiguous sections were processed for IHC staining with LC3B antibody at a 1:250 dilution. The sections were then incubated with the Supersensitive 1-step polymer-HRP detection system (Biogenex). Immunostaining was visualized with 3,3-diaminobenzidine (DAB, Biogenex) substrate. After counterstaining in hematoxylin, the sections were mounted and examined. The studies on clinical samples were conducted in accordance with ethical guidelines of the Institutional Review Board of Tongji University.

RT² Profiler Human Autophagy PCR Array (PAHS084ZE), RNeasy mini kit, RT² First Strand Kit, primers for qPCR validation, including BECN (PPH05670B), MAP1LC3A (PPH19436A), ATG4B (PPH15916A), DRAM1 (PPH19768F), CTSD (PPH00112F) and RPLPO (PPH21138F) were purchased from Qiagen-Sabiosciences. 3,3′-diaminobenzidine (DAB) was from BioGenex. TRIzol® reagent, 4-12% NuPAGE gels and Hanks balanced salt solution (HBSS) were from Invitrogen. Chloroquine (CQ), 4′,6-diamidino-2-phenylindole (DAPI), and all other chemicals used were from Sigma. Antibodies used in this study are listed in Suppl Table 5.

Paraffin-embedded sections were deparaffinized in xylene and rehydrated in descending concentrations of ethanol. After being washed in PBS, antigen retrieval was performed in boiling 10 mM sodium citrate solution (pH 6.0) containing 0.05% Tween-20 for 20 min. Sections were subsequently incubated in 3% H₂O₂ for 10 min and then in Rodent Block M (Biocare Medical, Concord, CA, USA) at RT. After 1 h, the sections were incubated with primary antibodies diluted in Tris-HCl buffer containing 0.1% Tween and 0.015 M NaN₃ (Dako Antibody Diluent; Dako North America) for 16 h at 4 °C. Rabbit, mouse, and rat IgG were used as negative controls. Following three washes in PBS containing 0.5% Tween-20 (PBST) for 10 min, the appropriate horseradish peroxidase (HRP)-labeled secondary antibodies were applied for 1 h at RT. To detect mouse or rabbit primary antibodies versus rat or goat primary antibodies, the Super Sensitive Polymer-HRP IHC Detection System (BioGenex, San Ramon, CA, USA) or Histofine Simple Stain MAX PO Kits (Nichirei, Tokyo, Japan) were used, respectively, with 3,3′-diaminobenzidine (DAB) as the chromogen (BioGenex). Nuclei were counterstained with hematoxylin. The sections were dehydrated and mounted with coverslips (Malinol NX; Muto Pure Chemicals).