Anti desmin

Nycodenz-isolated HSCs cultured for 1 day or for three passages were washed with PBS and fixed for 10 min with 4 % buffered formaldehyde (Merck, Darmstadt, Germany). Following permeabilization with 0.1 % Triton X-100 (in PBS containing 1 % bovine serum albumin), cells were incubated overnight with anti-αSMA (1/1000) (Sigma). Primary antibody binding was visualized using an Alexa488-labeled secondary antibody (1/200) (Invitrogen, Eugene, OR, USA). Images were taken with an AxioCam MRc5 digital camera (Carl Zeiss). NCAM1, nestin, and desmin stainings were performed as described previously [59 ]. Briefly, the cells were fixed, endogenous peroxidase was eliminated, and cells were permeabilized using PBS containing 1 % Triton X-100 (Sigma). Thereafter, the cells were blocked by 1-h incubation in PBS containing 1 % bovine serum albumin (Sigma), incubated with primary anti-NCAM1 (1/100) (Abcam), anti-Nestin (1/1000) (Abcam), or anti-Desmin (1/50) (Abcam) for 1 h, washed and incubated with secondary antibody (Envision, Dako) for 30 min. Detection was performed after 5 min incubation with liquid DAB and substrate chromogen (Dako). Counterstaining was performed using Mayer’s hematoxylin for 10 min. Preparations were then mounted for microscopic analysis (DMIL, Leica, Belgium).

After de-paraffinization, the sections were incubated with a 3% H₂O₂ solution to block endogenous peroxidases. Antigen retrieval was carried out using 0.1 M sodium citrate (pH 6.0) for 60 min. Sections were incubated with anti-α-SMA (1:100; BIOSS), anti-desmin (1:100; Abcam), anti-TGF-β1 (1:100; Santa Cruz Biotechnology, Inc.), anti-NF-κB p65 (1:100; Affinity Biosciences) or anti-SREBP-1 (1:100; Santa Cruz Biotechnology, Inc.) antibodies overnight at 4 °C, and a horseradish peroxidase-conjugated secondary antibody and diaminobenzidine substrate were added sequentially. Following hematoxylin counterstaining and dehydration, the sections were mounted and observed under a Leica DM4000B photomicroscope (Leica Microsystems, Inc.).

For IF, the colon was removed, faeces were gently washed out with cold PBS and the tissue was immediately frozen on dry ice in Tissue-Tek O.C.T. compound and stored at −80 °C. Sections were cut using a cryotome at 20–100 μm and incubated for 30 min in PBS with 0.5% Triton X-100 (Sigma-Aldrich; T8787) and 0.1% TOPRO-3 (Invitrogen; T3605). Slides were mounted between two coverslips (Menzel, Nr. 1.5 (0.16–0.19 mm)) and scanned using a Zeiss LSM710 confocal microscope in single-photon mode. For antibody staining, 20–100 μm sections were cut in a cryotome and incubated overnight in antibody diluted in PBS plus 0.5% Triton-X-100, 0.5% BSA and 0.5% dimethyl sulfoxide at 4 °C. A species-appropriate secondary antibody (1:250 dilution) was chosen from Alexa Fluor dyes (Invitrogen) using an appropriate wavelength depending on the primary antibody and the presence of endogenous fluorophores. The antibodies and dilutions were as follows: anti-vimentin (Santa Cruz, sc7557, 1:500); anti-desmin (Abcam, ab8592, 1:500); anti-α-sma (Abcam, ab5694, 1:500); anti-collagen1 (Abcam, ab34710, 1:200); anti-β-catenin (Cell Signalling, #9582, 1:100); anti-Ita6 (CD49f, BD Pharmigen, # 555734, 1:100).

All procedures have been described in detail previously [20 (link)]. Briefly, after an overdose of anesthetics, animals were transcardially perfused with physiological saline followed by 4 % PFA in 0.1 M phosphate buffer, pH 7.4. Brains were dissected from the skulls and postfixed overnight. Before sectioning from a dry ice-cooled copper block on a sliding microtome (Leica), brains were transferred to 30 % sucrose in 0.1 M phosphate buffer, pH 7.4. Brains were cut in the coronal plane in 40 μm thick sections. Sections were stained free floating. Immunohistochemistry followed the peroxidase method with a biotinylated secondary antibody (1:500; Jackson ImmunoResearch Laboratories, West Grove, PA), ABC Elite reagent (Vector Laboratories, Burlingame, CA) and diaminobenzidine (DAB; Sigma) as chromogen. For immunofluorescence, FITC- and RhodX-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) were all used at a concentration of 1:250. Primary antibodies were applied in the following concentrations: anti-NeuN (mouse, 1:100; Chemicon, Temecula, CA), anti-desmin (mouse, 1:100, #6322, Abcam), anti-Glucose Transporter-1 (anti-Glut-1; rabbit, 1:100, #400060, Calbiochem).

C2C12 cells were grown in culture medium for 5 days before transferring to differentiation medium (High-glucose DMEM supplemented with 2% horse serum and 1% P/S) for 7 days. The myotubes were treated with 3.7% paraformaldehyde for 20 min, then permeabilized using 0.3% triton X-100 in 5 min. Samples were blocked from binding to nonspecific antibodies using 5% rabbit serum in DPBS for 30 min, then washed with DPBS for 5 min. Primary antibody incubation of monoclonal antibodies was carried out to achieve optimal concentrations in a humidified chamber at 4 °C (1/100 dilution of anti-Desmin (Abcam, Cambridge, UK)) overnight. Sections were washed with 0.05% tween solution and incubated with Alexa Fluor 488-conjugated rabbit anti-mouse IgG containing 5% rabbit serum. 4′,6-diamidino-2-phenylindole was used to perform counterstaining of cell nuclei.

Formalin fixed paraffin-embedded (FFPE) tumour samples were sectioned and stained with haematoxylin and eosin (H&E) before pathological review and determination of tumour purity. Sections were also stained with anti-smooth muscle actin (Clone E184, Abcam), anti-desmin (Polyclonal, Abcam), anti-Ki67 (MIB-1, Dako), and anti-PAX8 (polyclonal, Proteintech) using the Ventana BenchMark Ultra fully automated staining instrument (Roche Diagnostics, USA). H&E and IHC slides were digitally scanned (20 × magnification) using the Pannoramic 1000 scanner (3DHISTECH Ltd.). High-definition images were uploaded into CaseCenter (3DHISTECH Ltd.), and images were processed using Adobe Illustrator.