Lysozyme

Small intestine and colon were isolated from mice and flushed with PBS followed by 4% paraformaldehyde (pH 7.4). Tissue was opened longitudinally, washed briefly in PBS, and incubated in 4% paraformaldehyde (pH 7.4) at 4°C overnight. A small, square piece of tissue was excised with a scalpel, embedded in 3% Low Melt Temperature Agarose, and sliced into 200 μm thick sections with a Vibratome (Leica, Wetzlar, Germany). Tissue sections were permeabilised for 2 h in 2% Triton-X100 (this and all subsequent steps at 4°C), blocked for 2 h (1% BSA, 3% normal goat serum, 0.2% Triton-X100 in PBS), and incubated in primary antibodies for 3 d (Lysozyme and β4 Integrin as described above, Muc2 [1:200, Santa Cruz Biotech, Santa Cruz, CA]), washed in Working Buffer (5x in 1 h), incubated in secondary antibodies along with Phalloidin and Hoechst (as described above) and Rhodamine-labelled UEA-I (1:200, Vector Labs, Peterborough, UK), washed in Working Buffer (5x in 1 h), and mounted in ProLong Gold antifade (Molecular Probes). Sections were mounted on coverslips between 2x 120 μm spacers to preserve tissue structure.

The following antibodies were used for western immunoblotting: PHB1 (70R-5543, Fitzgerald), PHB2 (14085, Cell Signaling), LC3 (L7543, Sigma), p62 (5114, Cell Signaling), Tim50 (sc-515268, Santa Cruz), CoxIV (4850, Cell Signaling), Nix (12396, Cell Signaling), FundC1 (ABC506, Millipore), Bnip3 (3769, Cell Signaling), Optineurin (100000, Cayman Chemical), NDP52 (H000 10241-B01P, Abnova), GST (sc-138, Santa Cruz), Parkin (MAB5512, Millipore), GFP (2956, Cell Signaling), Cytochrome C (4280, Cell Signaling), β-actin (A1978, AC-15, Sigma-Aldrich), β-tubulin (T4026, Sigma Aldrich). Antibodies were validated by western blot using the respective recombinant protein as positive control. The following antibodies were used for immunostaining: LC3 (L7543, Sigma), Lysozyme (sc27956, Santa Cruz), Muc2 (ab134119, EPR6145, Abcam), CoxIV (4850, Cell Signaling), PHB1 (70R-5543, Fitzgerald), and Nix (12396, Cell Signaling). Isotype controls were included to validate immunostaining.