Donkey anti rabbit hrpconjugate secondary antibody

THP-1 cells were infected at an MOI of 10. At 24 hours after infection,
cells were lysed in 0.5% Triton X-100 (Sigma) plus protease inhibitors
(Roche) and samples were filtered twice with a 0.22 µm microcentrifuge
filter (Millipore). Blotting was performed with rabbit anti-HO1 polyclonal
antibody (1:1000, ADI-SPA-896-F, Enzo Life Sciences) or mouse anti-β
actin antibody (1:1000, Santa Cruz, sc-47778) and donkey anti-rabbit-HRP
conjugate secondary antibody (1:5000, sc-2305, Santa Cruz) or goat
anti-mouse-HRP conjugate (1:5000, sc-2005, Santa Cruz), respectively.
Supersignal West Femto Chemiluminescent Substrate (Pierce) was used for signal
detection.

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.