Cd117 c kit

Prior to immunolabeling, slides were boiled in citrate buffer for antigen-retrieval, then washed in phosphate-buffered saline (PBS) and permeabilized in PBS containing 1% bovine serum albumin (BSA) for 30 min. Primary antibodies (CD117/c-kit, Dako, USA) were applied in a 1:100 dilution for 1 h at 4 °C. A HRP-labelled polymer conjugated with secondary antibodies and 3,3′-diaminobenzidine (DAB)+ substrate-chromogen [EnVision+ System-HRP (DAB), Dako, USA] were used to detect the primary immune reaction. Following incubation steps, slides were rinsed three times in PBS. After labeling, the coverslips were mounted with Canadian balsam. The slides were examined under a light microscope BX-61 (Olympus, Poland) using MicroImage, v. 4.0 software (Olympus, Poland). Negative controls were prepared following the same protocol, but omitting the primary antibodies.

Mice were euthanized by inhalation of a 5% CO₂ / 95% O₂ followed by 100% CO₂ for 4 min, and transcardially perfused with PBS to remove blood. Human and mouse formalin-fixed paraffin-embedded 5-μm-thick small intestine tissue sections were routinely stained for Hematoxylin-Eosin and Phosphotungstic acid-hematoxylin and immunostained as described [18 (link)] against smooth muscle actin (SMA, Dako, 1:200), CD117/c-kit (Dako, 1:50), calretinin (Dako, 1:200), NeuN (Millipore, 1:100), CD3 (Dako, 1:250), CD8 (Dako, 1:50) and glial fibrillary acidic protein (GFAP, Dako, 1:300). Immunoreactivity was detected using 3,3'-Diaminobenzidin or Liquid Permanent Red as chromogen. Pictures were taken with a Leica DM3000 microscope. Muscle thickness was measured on transversally cut intestinal sections (N = 40 for mice, N ≥ 25 for humans). Quantification of muscle wall thickness and ganglion cell density was performed blind to the genotype with ImageJ.