Cell imaging station

Femurs with drill hole injury were dissected free of connective tissues, fixed in 4% buffered formalin, decalcified in 1% EDTA and embedded in paraffin. Transverse sections of 5μm were then cut from each sample. Sections were initially deparaffinized using xylene, rehydrated through an ethanol gradient and permeabilized with 0.1% Triton X-100 followed by blocking with 1% BSA. These were then incubated with β-catenin antibody diluted in 0.5% BSA (1:100) at 4°C overnight. After washings in 1× PBS, sections were incubated with Alexaflour 488 goat anti-rabbit (1:500). Sections were then washed with PBS and incubated with ALP antibody (1:100) diluted in PBS containing 0.5% BSA, overnight at 4°C under humid conditions. Sections were again washed with PBS and incubated with fluorescent Alexa Fluor-647 donkey anti-goat IgG (H + L) (1:500 dilution in PBS) (Molecular Probes, Carlsbad, CA,USA) at room temperature for 1 hour. Sections were counter stained with DAPI for 15 minutes in dark. After 15 min incubation slides were rinsed with PBS and mounted with antifade mounting media ((life technologies, Carlsbad, CA, USA). Sections were visualized under Cell Imaging Station (life technologies, Carlsbad, CA, USA).

Live/dead assay for the Mm–infected RAW264.7 cells–RAW264.7 cells (5 × 10⁵) were infected with the indicated Mm strains at an MOI of 20 for 2 h at 37 °C, followed by another 3 h incubation after washing off the free bacteria. The infected cells were stained with ethidium homodimer and calcein-AM (Life Technologies) at 37 °C for 1 h, and images were taken in a Floid Cell Imaging Station. The relative cytotoxicity was quantified by enumeration of dead cells in at least 6 random fields for each infection.
Crystal violet uptake assay for the Mtb-infected THP-1 cells–The PMA-differentiated THP-1 cells (5 × 10⁵) were infected with the indicated Mtb strains at an MOI of 10 for 72 h at 37 °C and fixed with 4% PFA for 30 min, and then stained with 0.1% aqueous crystal violet for 30 min with gentle shaking. The excessive dye was washed off with water. The plates were allowed to air dry and the stained cells were lysed in 0.2% Triton X-100 for 30 min at RT with gentle shaking. The supernatant was transferred into a 96-well plate for OD₆₀₀ measurement.

The sections were incubated in blocking solution for 1 h at room temperature. The incubation with the primary antibodies at a dilution of 1: 500 [(anti-P2Y2 (sc-518121], anti-TLR4 (sc-518121) and anti-NE (sc-55549) was carried out overnight at 4 °C. A 1:200 dilution of the secondary antibody m-IgGκBP (sc-516141) was used for NE and P2Y2; while for TLR4 a 1:400 dilution of mouse anti-rabbit IgG (sc-3753) with overnight incubation at 4 °C was done (all from Santa Cruz Biotechnology, CA, USA). Nuclei were labeled with DAPI. Observation and photographs for fluorescence images were obtained with a Cell Imaging Station (Life Technologies, Carlsbad, CA, USA).