Cyanine 3 tyramide

Sections from paraffin-embedded mouse and human lung were deparaffinized
in xylene, subjected to heat-mediated antigen-retrieval in 1 mM EDTA with
0.05% Tween 20 (pH 8.0), endogenous peroxide activity was quenched in
2.5% hydrogen peroxide solution in methanol, slides were permeabilized
in 0.1% Triton-100 (Sigma) and blocked in SuperBlock Blocking Buffer
(Thermo Scientific). For immunohistochemistry, sections were stained with rabbit
anti-HO1 (1:100, ADI-SPA-896-F, Enzo Life Sciences), rabbit anti-HA (1:50,
sc-805 Santa Cruz), or mouse anti-CD68 (1:100, ab955 Abcam). HRP-conjugated
secondary antibodies were obtained from Jackson Immunochemicals and used at
1:250. Staining was amplified with AB reagent (Vectastain) and detected using
DAB reagent (Thermo Scientific). Images were acquired using a Zeiss Axioplan 2
microscope. For immunofluorescence, HO1 was identified using rabbit anti-HO1
(1:100) and a donkey anti-rabbit-HRP conjugate secondary antibody (1:500, Santa
Cruz) followed by amplification with Cyanine 3 tyramide (1:100, Perkin Elmer).
Mtb was identified using guinea pig anti-Mtb (1:25, NR-13818, NR-13823 BEI) and
an Alexa 488 conjugated donkey anti-guinea pig secondary antibody (1:100,
706-545-148 Jackson Immunochemicals). All commercial antibodies were prepared
without Freund’s adjuvant. Images were acquired using a Leica TCS SP5
confocal microscope.

Riboflavin, Safranin-O, Fast Green FCF, dimethyl sulfoxide (Sigma-Aldrich); tyramine‐substituted sodium hyaluronate (873 kDa, 5.5 % substitution, w/w; LifeCore Biomedical); 1× phosphate buffered saline (PBS) and Dublecco’s Modified Eagle Medium (DMEM, LifeTechnologies); fetal bovine serum (FBS, Atlanta Biologicals); 10% formalin, xylene (Pharmco-Aaper); paraffin (Electron Microscopy Sciences); cyanine-3 tyramide (Perkin Elmer); LIVE/DEAD® Viability/Cytotoxicity Kit for mammalian cells and Penicillin-Streptomycin (ThermoFisher Scientific). All chemicals were used without further purification. Immature bovine knees were obtained from a local abattoir.

U937 or THP-1 cells were differentiated as described above. Cells were
infected at an MOI of 5 and 24 hours after infection, cells were washed with PBS
and fixed in 4% paraformaldehyde (Alfa Aesar). Cells were permeabilized
with 0.1% Triton X-100 and blocked with SuperBlock Blocking Buffer
(Thermo Scientific). HO1 was identified using rabbit anti-HO1 (1:100) and an
HRP-conjugated donkey anti-rabbit secondary (1:500, Santa Cruz) followed by
amplification with Cyanine 3 tyramide (1:100, Perkin Elmer). Mtb expressing GFP
were used for infections in THP1 and U937 cells. Images were acquired as
z-stacks using a Zeiss Axioplan 2 microscope and were deconvoluted using Imaris
and Autoquant softwares. Additional antibodies used to identify the location of
HO1 positive bacteria were mouse anti-LAMP1 (lysosome, 1:100, sc-20011, Santa
Cruz), mouse anti-Rab7 (late endosome, 1:100, ab50533, Abcam), mouse anti-SQSTM1
(for p62/sequestosome, 1:2000, H00008878-MO1, AbNova), and mouse anti-Sec22B
(ER-Golgi intermediate compartment, 1:50, sc-101267, Santa Cruz).