Immunohistochemical Analysis of Panx-1 in Mouse Colon

Sections (4 µm thick) were prepared from paraffin-embedded mouse colon and cecum tissues. After deparaffinization, antigens were recovered by incubating the slides in citrate buffer (pH 9.0) for 20 min in PT link tank (DAKO). Endogenous peroxidase was blocked with 3% H2O2 for 30 min to reduce nonspecific binding. The sections were then incubated with an Panx-1 antibody (R&D system) for 3 hours. The sections were then incubated for 30 min with polymer (K4061, Dako). The antibody binding sites were visualized by incubating the samples with diaminobenzidine–H2O2 (DAB, Dako) solution. Sections incubated with antibody diluent without a primary antibody were used as negative controls. Immunohistochemical images were captured using a light microscope coupled to a camera with a LAZ 3.5 acquisition system (LEICA DM1000, Germany). To quantify the positive immunostaining area for Panx-1, the Adobe Photoshop 8.0 program was used to obtain the total tissue and the positive area. The percentage of immunopositive area was calculated by dividing the number of DAB-positive pixels (immunostaining-positive pixels) by the number of pixels per total tissue image and multiplying the result by 100, as previously described (36 (link)).