Aquatex

Tissue microarrays (#MC245c and MC246b, amsbio) were baked for 1 h at 60°C before deparaffinization and rehydration through xylene, ethanol, and deionized (dd) H₂O. Tris/EDTA buffer, pH 9.0 at 95°C for 30 min was used for antigen retrieval. Specific binding of primary rabbit polyclonal anti-IRF6 antibody (NBP2-49383, Novus Biologicals, Centennial, CO, USA) was visualized with the ImmPACT DAB Peroxidase Substrate (Vector Laboratories, Newark, CA, USA). Nuclei were counterstained with hematoxylin and slides mounted with Aquatex (Sigma-Aldrich, St. Louis, MO, USA).

The liver tissue was sectioned at −20 °C on a cryostat microtome after frozen fixation via OCT (10 μm thick, Cryostar NX70, Fisher Scientific, Waltham, MA, USA).
For hematoxylin–eosin (HE) staining, cryosections were fixed in acetone for 3 s and dried at 37 °C. After an hour of drying, the cryosections were stained in Harris’ hematoxylin solution for 2 min, washed in tap water for 3 min, bleached in 1% acid alcohol for 2 s, and then again washed in tap water for 3 min. Then, the cryosections were counterstained in eosin solution for 1 min, washed in tap water for 3 s, in 80% ethanol, in 100% ethanol, and finally mounted with Eukitt medium (Orsatec, Germany).
Cryosections were rehydrated in PBS for 2 min, stained in Oil Red O solution (Sigma-Aldrich, O0625, Burlington, MA, USA) for 3 min, washed in isopropanol (60%) for 30 s, and subsequently washed in deionized water for 1 min. The slides were counterstained by dipping them in Harris’ hematoxylin for 3 min, washed in tap water for 2 min, and mounted in aqueous medium with Aquatex (Sigma-Aldrich, 108635). The stained slides were blinded and captured using a Zeiss Apotome.2 microscope (CTK Instruments, Carlsbad, CA, USA), and lipid accumulation was quantified with Adobe Photoshop (Adobe Systems, San Jose, CA, USA).

Aquatex, DNA oligonucleotide, duplex siRNA oligonucleotide for RNA interference (RNAi), 4′,6-diamidino-2-phenyl-indol-dihydrochlorid, 2-(4-amidinophenyl)-6-indolcarbamidin (DAPI), -dihydrochloridas (D9542-5MG), as well as reagents for molecular bacteriology were purchased from Sigma (Sigma Aldrich, Merck, Darmstadt, Germany). Deoxynucleotide triphosphates of PCR grade were obtained from Roche (Roche Diagnostics, Manheim, Germany). Calcium and magnesium free-Dulbecco’s phosphate buffered saline (PBS), Dulbecco’s modified Eagle medium (DMEM) (high glucose), DMEM/F12, OptiMEM, penicillin/streptomycin (5000 U/ml), sodium pyruvate (100 mM), and Trypsin EDTA (0.05%) were purchased from Gibco (Gibco, Thermo Fisher Scientific Darmstadt, Germany). Fetal calf serum (FCS), was from Biochrom (Biochrom KG, Berlin, Germany). Growth factors and cytokines were obtained from Peprotech (Peprotech, Hamburg, Germany). Lipofectamine 2000 was acquired from Invitrogen (Invitrogen, Thermo Fisher Scientific). Random hexameric primer was obtained from Qiagen (79236; QIAGEN, Hilden Germany), and 2 × qPCRBIO Probe mix Lo-ROX was obtained from Nippon Genetics (NIPPON Genetics Düren, Germany). PKM2 activator thieno-[3,2-b]pyrrole[3,2-d]pyridazinone NCGC00186528 (TEPP-46;ML265, PubChem CID 44246499) [28 (link)] was obtained from MedChemEexpress (Hoelzel-Biotech, Cologne, Germany).

From each formalin-fixed and paraffin-embedded sample, 5 μm-thick sections were cut. Histological sections were deparaffinized and rehydrated using graded alcohols, and endogenous peroxidase activity was blocked for 30 min in a 0.3% H₂O₂ methanol solution. Heat-induced antigen retrieval was performed using a pressure cooker for 20 min in a citrate buffer (pH 6.0). After washing in Tris-buffer, protein blocking was performed using normal horse serum for 30 min at 25°C. Sections were then incubated with a mouse anti-human Ki-67 primary antibody (MIB-1; Dako, CA, USA) diluted 1:600 in Tris buffer for 18 h at 4°C. After rinsing sections in Tris buffer, slides were incubated with a biotinylated horse anti-mouse secondary antibody (Vector Laboratories, CA, USA) for 30 min at 25°C. Immunohistochemical signals were detected using an avidin-biotin system (Vector Laboratories, CA, USA) and 3-amino-9-ethylcarbazole (AEC) substrate-chromogen kit (Vector Laboratories, CA, USA). Sections were counterstained with Harris hematoxylin and mounted using an aqueous mounting medium (Aquatex, Sigma-Aldrich, MO, USA). The Ki-67 value was expressed as the percentage of positively stained cells, calculated by counting 1,000 cells per section (400× magnification).

Lung specimens were washed with phosphate-buffered saline through the pulmonary artery, fixed in 4% formalin and embedded in paraffin. Tissue sections (4 μM) were stained with hematoxylin and eosin (H&E) or with Elastica van Gieson for morphometric analysis. For each artery, the percentage of medial wall thickness was measured at the two ends of the shortest external diameter of the distal pulmonary artery, and the average was taken as previously described [51 (link)]. Immunohistochemistry staining of paraffin-embedded lung sections was stained with anti-HIF-1α antibody followed by HRP-conjugated goat anti-mouse IgG2b (Santa Cruz). The immunostained samples mounted with Aquatex (Sigma-Aldrich) were examined under a LEICA microscopy (Leica, Wetzlar, Germany). Images were quantified with an image analyzer (LEICA Q550 IWB).

Mice were anesthetized in a hermetic cage, ventilated with gas mixture of 4% isoflurane (Aerrane, CSP, Cournon, France) and oxygen. Gastrocnemius and quadriceps muscles were snap-frozen for biochemistry or fixed in isopentane for histological analysis. Mice were euthanized by cervical dislocation and exsanguinated. The gastrocnemius tissue was frozen-fixed in OCT and sectioned at −20 °C on a cryostat microtome (10-μm thick, Cryostar NX70, Fisher Scientific, Waltham, MA, USA). For NADH-tetrazolium reductase staining, cryosections were incubated in 0.2 M Tris-HCl pH 7.4, containing 1.5 mM NADH and 1.5 mM nitrobluetetrazolium (NBT) for 15 min at 55 °C. Then, they were washed with three exchanges of deionized H₂O. Unbound NBT was removed from the sections with three exchanges each of 30, 60 and 90% acetone solutions in increasing and then decreasing concentration. Finally, the sections were washed several times with deionized water and mounted with aqueous mounting medium (Aquatex, SIGMA-ALDRICH, 108635) [19 (link)].