Anti lama2

Cells and skeletal muscle tissues were fixed with 4% paraformaldehyde in PBS for 10 min at 4 °C, before embedding in Frozen Section Compound (Leica Microsystems) for cryosections. Fixed samples were incubated with anti-PAX7 (DSHB; diluted 1/100), anti-MYOG (DAKO; diluted 1/100), anti-DYSTROPHIN (DAKO; diluted 1/100), anti-MyHC (MF20, R&D; diluted 1/ 200), anti-LAMA2 (Enzo Life Sciences, Farmingdale, NY, USA; diluted 1/500), anti-hLMNA (Abcam; diluted 1/200), and anti-FSP1 (Abcam; diluted 1/200) antibodies in 5% of BlockingOne (Nacalai) for overnight at 4 °C. After three washes with 0.1% of Tween20 in PBS, cells were incubated with Alexa488, Alexa594, or Alexa647-conjugated secondary antibodies (Molecular Probes; diluted 1/500). Cells were washed and mounted in SlowFade Diamond Antifade Mountant with DAPI (Molecular Probes, Eugene, OR, USA). Images were collected and processed to change original fluorescent colors when appropriate on the software of BZX-710 (Keyence, Osaka, Japan). For quantitation of cultured cells, numbers of dishes were analyzed from triplicate experiments.

Fixed human cadaver pancreatic tissue (Prodo) was incubated in 30% sucrose solution followed by embedding in OCT (TissueTek), cryopreservation, and sectioning. Dissected mouse pancreatic tissues were fixed with Z-fix (Anatech), incubated in 30% sucrose overnight, and cryopreserved. Tissue sections were stained with primary antibodies, anti-mouse vimentin (1 : 200, Sigma), anti-human vimentin (1 : 50, Calbiochem), anti-YFP/GFP (1 : 500, Abcam), anti-Galectin-1 (1 : 50, RnD Systems), anti-LAMA2 (1 : 200, Enzo), and anti-PDX1 (1 : 200, RnD Systems), followed by staining with AlexaFluor tagged secondary antibodies (1 : 500, Invitrogen) and mounting Vectashield media (Vector). Nuclei were visualized with DAPI. Images were acquired using a Leica SP5 microscope or an InCell Analyzer 2000 for quantification (GE Healthcare). 16 fields from each condition were randomly selected by the InCell Analyzer and imaged for quantification. PDX1 positive nuclei over total nuclei were determined using InCell Developer software (GE Healthcare).