Primary Ab binding was revealed using horseradish peroxidase-conjugated goat anti-rabbit Ab (Jackson Immunoresearch, West Grove, PA, USA) and the ECL chemiluminescence detection system (Pierce, Rockford, IL, USA). Quantification analyses were performed by LAS3000 Image System (Fuji, Milano, Italy) and ImageQuant software (GE Healthcare, Milano, Italy). Transverse and longitudinal muscle cryostat sections (6 µm) were thaw-mounted on

to glass slides pretreated with 3% EDTA to prevent contracture artefacts, and processed for indirect IF as previously described.[36]. In brief, after fixation in acetone for 10 min at 4°C, sections were incubated for 60 min with the K20 Ab (1:40/1:100), rinsed in phosphate buffered saline (PBS) and incubated for 30 min with a FITC-conjugated goat anti-rabbit (Sigma, selleck screening library Milano, Italy, 1:50) Ab. Negative control sections were

incubated with non-immune serum instead of primary Ab. In order to evaluate a possible co-localization of ZNF9 with cellular organelles or cytoskeletal components, double-labelling experiments were conducted using specific markers for the sarcoplasmic reticulum, mitochondria, ribosomes, intermediate filaments and sarcomeric structures. To this purpose, Smad inhibitor an anti-sarcoplasmic reticulum calcium ATPase (SERCA1) monoclonal Ab (mAb) (Biomol, Hamburg, Germany, 1:200), an anti-S6 ribosomal protein mAb (Cell Signaling, Danvers, MA, USA, 1:100), IKBKE an anti-desmin mAb (Sigma, Milano, Italy, 1:100) and two mAbs recognizing the T11 and T12 epitopes of titin (Sigma, Milano, Italy, 1:20) were used. Mitochondria were labelled with 100 nM Mitotracker Green FM (Molecular Probes, Milano, Italy) for 45 min. To analyse ZNF9 distribution among different myofibre types, transverse sections double-labelled for ZNF9 and SERCA1 (specific for fast myofibres) were observed. In addition, serial sections labelled for ZNF9 or routinely stained with the histoenzymatic reaction for myofibrillar ATPase (pH 4.3) were compared. For myelin counterstaining of

intramuscular nerve twigs, the lipophilic carbocyanine DiOC6(3) dye (Molecular Probes, Milano, Italy) was used at a concentration of 1 µg/ml for 5 s (R. Massa, pers. obs.). Brains were sectioned frozen on a sliding microtome at 40-µm thickness and incubated overnight with K20 Ab (1:100), rinsed in PBS and then incubated for 90 min with a FITC-conjugated goat anti-rabbit (1:50) Ab. Negative control sections were incubated with non-immune serum instead of primary Ab. All sections were mounted with anti-fading medium and routinely examined and photographed with an Olympus BX51 microscope (Olympus, Milano, Italy) by epifluorescent excitation. Confocal analysis was carried out with a Zeiss LSM 510 system (Zeiss, Milano, Italy), equipped with 40 × 1.00–0.5 and 100 × 1.3–0.6 oil immersion lenses. Serial optical sections, 0.