Slowfade mounting medium

Paraffin-embedded tissue sections were deparaffinized, and antigen retrieval was performed using boiling 0.01M citrate buffer (pH 6) for 20 min. Sections were blocked with 5% goat serum in Dako serum-free blocking reagent for 30 min. For α-smooth muscle actin (α-SMA) staining, sections were incubated overnight at 4°C with Cy3-conjugated α-SMA antibody (1:400)(Sigma-Aldrich, Oakville, Ontario, Canada). After washing with PBS-0.1% Tween, sections were counterstained with DAPI and mounted with Slow Fade mounting medium (Invitrogen, Waltham, MA, USA). For CD3/B220 staining, the sections were incubated overnight at 4°C with CD3 antibody (1:200)(DAKO, Mississauga, Ontario, Canada) and Biotin conjugated B220 antibody (1:250)(BD, Mississauga, Ontario, Canada). After washing, sections were incubated with goat anti-rabbit IgG Alexa Fluor^® 555 and Alexa Fluor^® 488 streptavidin (Molecular Probes, Eugene, OR) at the dilution of 1:400. Antibodies for myeloperoxidase (MPO) (1:400) and F4/80 (1:500)(Abcam, Cambridge, MA, USA) were used for neutrophil and macrophage staining, respectively. Goat anti-rabbit IgG Alexa Fluor^® 555 (Molecular Probes, Eugene, OR, USA) were used as the secondary antibody at the dilution of 1:400. Then the sections were counterstained with DAPI and mounted.

Paraffin tissues were embedded and sectioned at 5 μM and dewaxed in xylene and rehydrated in alcohol with citrate antigen retrieval. Standard Mayer’s hematoxylin and eosin (H&E) was performed. The following antibodies were used: Krt8/18 (Fitzgerald 20R-CP004 1:500) and Krt5 (BioLegend 905,501 1:500). Paraffin-derived sections were counterstained with hematoxylin (Vector Labs) and mounted with Cytoseal. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 12% Fraction-V BSA (Pierce) and slides mounted in SlowFade mounting medium containing DAPI (Invitrogen). All fluorescent secondary antibodies (Krt5: Goat Anti-Rabbit A488 A-11034 (K8/18: Goat Anti-Guinea-Pig A594 A-11076, Thermo Fisher) were highly cross-adsorbed and used at a dilution of 1:200 for 20 min. Nuclei were stained with SlowFade Gold with DAPI (S36939 Thermo Fisher).

2 x 10⁶ PF T. brucei cells were incubated in 1 mL GF-SDM-79 growth media supplemented with/without inhibitors for 2 h. B-THP-Ts were used at 40 μM, AA and OA at 2 μM, DNP at 1 mM and SHAM at 100 μM. MitoTracker Red CMXRos and MitoTracker Green FM were added to 100 nM and 1 μM respectively and incubated for a further 10 min. Cells were washed in growth medium and the MitoTracker reagents chased into the mitochondrion for 10 min in fresh growth medium supplemented with inhibitors. Cells were washed again in growth medium to remove unincorporated MitoTracker reagents, pelleted, suspended in 100 μL 4% PFA in PBS, and fixed at RT in the dark for 10 min. Fixed cells were pelleted and suspended in 100 μl PBS. Cells were transferred to poly-lysine-coated slides and mounted with DAPI-containing SlowFade mounting medium (Invitrogen). Fluorescence images were collected with a DeltaVision microscope (as detailed above) to show MitoTracker localisation and comparative intensity.

The muscle samples were fixed in a 4% formaldehyde solution at room temperature for 30 min. Subsequently, the samples were rinsed with PBST (PBS with 0.1% Triton X-100) for 4 times, 15 min each time. The samples were then treated with a blocking solution containing 5% BSA in PBS. Following this, the samples were incubated with primary antibodies overnight at 4 °C. Afterwards, the samples were washed again with PBST for 4 times, 15 min each time, and then incubated with secondary antibodies labeled with Alexa Fluor 568 or Alexa Fluor 488 (1:500 dilution, Invitrogen) for 2 h at room temperature. After washing with PBST for 4 times, 15 min each time, the samples were mounted using Slow-Fade mounting medium (Invitrogen) for imaging. The primary antibodies used in this study were chicken anti-GFP antibody (1:1000, Abcam, ab13970); mouse anti-HA antibody (1:1000, Santa Cruz, sc7392); mouse anti-Cnx99A antibody (1:100, DSHB). Representative images were shown for each genotype, with eight individual samples for each genotype analyzed. All tissue immunostaining images were obtained under identical gain settings as well as laser intensities.

Right #4 inguinal mammary glands were formalin fixed and paraffin embedded, and sections (5 μm) were stained with hematoxylin and eosin. In-situ terminal dUTP nick end-labeling (TUNEL) analysis was performed on paraffin-embedded sections using the ApopTag kit (Millipore). Immunohistochemistry (IHC) on paraffin-embedded sections was performed using the following antibodies as described previously [50 (link)]: ErbB3 (C-17; Santa Cruz Biotechnology), Ki67 (Santa Cruz Biotechnology), P-Akt S473 (Cell Signaling Technologies), P-STAT5A/B (Neomarkers), and P-ErbB4 Tyr1056 (Cell Signaling Technologies). Immunodetection was performed using the Vectastain kit (Vector Laboratories) according to the manufacturer’s instructions. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 12% Fraction-V BSA (Pierce) and slides were mounted in SlowFade mounting medium containing DAPI (Invitrogen). All fluorescent secondary antibodies were highly cross-adsorbed, produced in goat, and used at a dilution of 1:200 for 20 min (Molecular Probes). Primary antibodies used were CK5 (10956, 1:500; Covance/Biolegend), CK8/18 (1:500; Fitzgerald Industries International), and ErbB3 (c-17, 1:200; Santa Cruz Biotechnology).

Paraffin tissues were embedded and sectioned at 5μM and dewaxed in xylene and rehydrated in alcohol with citrate antigen retrieval as previously described [5 (link)]. Standard Mayer's hematoxylin and eosin (H&E) was performed. Cleaved Caspase-3 (Cell Signaling Cat#9661, 1:200), Vimentin (Covance Cat#PCK-594P 1:500), BrdU (BD Cat#563445 1:100). pSmad1/5 (Cell Signaling Cat#9516 1:200 ), Snail (Santa Cruz Cat#28199 1:200), Slug (Santa Cruz Cat#166476 1:100), Ecadherin (BD Cat#610181 1:200), K8/18 (Fitzgerald Cat#20R-CP004 1:500), K5 (Covance Cat#PRB160P 1:500), pSmad2 (Cell Signaling Cat#3101 1:500), CollagenIV (Abcam Cat#19808 1:500) and p63 (Santa Cruz Cat#8344 1:200). Paraffin derived sections were counterstained with hematoxylin (Vector Labs QS) and mounted with Cytoseal. Immunofluorescence staining was performed with primary and secondary antibodies diluted in 12% Fraction-V BSA (Pierce) and slides were mounted in SlowFade mounting medium containing DAPI (Invitrogen). All fluorescent secondary antibodies were highly cross-adsorbed, produced in goat and used at a dilution of 1:200 for 20 min (Molecular Probes). Quantification of IHC and IF was performed using NIH ImageJ (http://rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html) as previously described [43 (link)].