Semi-automatic counting of connexin 32s immunolocalized in cultured fetal rat hepatocytes using image processing

Connexin 32s (Cx32s) were immunolocalized in fetal rat hepatocytes and their distribution was determined qualitatively. We used image analysis using a quantitative index (QI) of Cx32 (QI Cx32) defined as the area of Cx32s/number of cells in cultured fetal rat hepatocytes. Hepatocytes from livers of fetal rats were separated by collagenase digestion and low centrifugation on gestational day 17. Cells were cultured for 3 days in dexamethasone (DEX)-supplemented medium (Dex0). The medium was replaced with fresh medium and cells were continuously cultured for 3 days with DEX or epidermal growth factor supplemented medium (Dex3 or EGF3). After culture termination, cells were fixed and stained using the fluorescein-labeled antibody method for Cx32s and diaminophenylindole staining for nuclei. Thirty pairs of histological images for Cx32s and nuclei, 180 images in total, were obtained from each condition. The QI Cx32 significantly increased from 284.1 ± 102.0 (mean and SD, n=26) of Dex0 to 428.9 ± 101.0 of Dex3 (n=28) (P<0.05, Kruskal-Wallis test, then Steel-Dwass test). The increase of QI Cx32 was compatible with the morphological observations. The image analysis processing time after preparation for 180 images was reduced from 8h needed for manual operations to 1 min using ImageJ software with our macro routine.