Mowiol

Adult muscles were dissected and embedded with OCT compound. 10 μm sections were obtained and fixed in 4% paraformaldehyde for 10 min. For PAX7 staining, antigen retrieval protocol (10 mM sodium citrate buffer, pH 6) was performed. Muscle sections were blocked with 10% goat serum/0.4% Triton/PBS for 1 h and incubated with primary antibody (anti-PAX7 in 1% BSA/0.04% Triton/PBS) overnight. The next day, sections were incubated with secondary antibody Alexa Fluor (1:300) and anti-LAMININ for 1 h and with Hoechst for 10 min. For DYSTROPHIN and F4/80 staining, muscle sections were permeabilized and blocked with 1% BSA/1% goat serum/0.025% Tween 20/0.2% (Triton X-100/PBS) for 1 h and incubated with primary antibody (anti-DYSTROPHIN) overnight. The following day, sections were incubated with secondary anti-rabbit Alexa Fluor antibody with or without Alexa Fluor-conjugated anti-F4/80 for 1 h and with Hoechst for 10 min. Slides were mounted with Mowiol (VWR) reagent.

BMDM were seeded in 24-well plates containing 0.01% polylysine solution (Sigma P4707) coated 12 mm cover glasses (Faust 6080181) at 2.5 x 10⁵ cells/well and rested overnight. Where indicated 100 ng/ml TNF (Peprotech, 315-01A) or 200 U/ml IFNγ was added to pre-activate the BMDM. Cells were infected with Lpn-GFP as described above at MOI 5 for 1 or 3 hours at 37°C, 5% CO₂, with the simultaneous addition of100 ng/ml TNF or 200 U/ml IFNγ where indicated. For the final 30 minutes of incubation 1μM lysotracker Red DND-99 (Life Technologies, L7528) and 0.5 μg/ml Cholera toxin B AF647 (CTB-AF647, Life Technologies, C34778) were added to the cells. Cells were then washed with 1 ml PBS, and cover glasses were then placed on parafilm, and fixed 5–10 min at RT with 200 μl 4% PFA in PBS. Cells were washed 3 times with 200 μl PBS, incubating 2 min after applying each wash. Cover glasses were dipped in dH₂O, blotted on paper towel to remove excess water and mounted on glass slides with cells facing downwards with 6 μl Mowiol (VWR, 475904–100). Z-stack images were acquired on a spinning-disk confocal microscope (Visitron confocal system) using a 100x objective, and analyzed with volocity software (PerkinElmer, Waltham, MA). To assess co-localization of L. pneumophila and lysosomes, at least 100 bacteria were scored per coverslip.

For MHC, ß-Galactosidase and ELMO staining, experiment was performed as previously described^{5 (link)}. Briefly, embryos were fixed in 4% paraformaldehyde for 1 h and incubated in 20% sucrose/PBS overnight. 10μm cryosections of embryos were obtained and antigen retrieval protocol (10 mM sodium citrate buffer, pH 6) was performed. Following blocking in 5% BSA/0.1% Tween-20/PBS for 1 h, embryo sections were incubated with primary antibody overnight. The next day, sections were incubated with secondary anti-mouse (MHC) or anti-rabbit (ß-Galactosidase and ELMO) antibody for 1 h and with Hoechst for 10 min. TUNEL kit was used to detect apoptotic cells, according to the manufacturer’s protocol. Anti-phospho-HISTONE-H3 antibody was used to detect mitotic cells. For MYOD and DESMIN staining, embryos were fixed in 4% paraformaldehyde for 5 min and incubated in 20% sucrose/PBS overnight. 10 μm sections were obtained from OCT: 20% sucrose/PBS (1:1) embedded embryos and permeabilized with 0.2% Triton/PBS. Following blocking in 1%BSA/PBS for 1 h, embryo sections were incubated with primary antibody (anti-MYOD or anti-DESMIN) overnight. The next day, sections were incubated with secondary antibody for 1 h and with Hoechst for 10 min. Slides were mounted with Mowiol (VWR) reagent.