Bx51tf fluorescence microscope

Approximately 10⁴ nthy-ori 3–1 cells were grown on coverslips and exposed to treatments. The fixed and permeabilized cells were blocked in 5% BSA and then incubated with a 1:100 dilution of primary Pax8 antibody (Abcam, San Francisco, CA, ab53490) and Dnmt3a antibody (Santa Cruz, CA, sc-20703) at 4 °C overnight. They were incubated with a 1:200 dilution of Alexa Fluor594 goat anti-mouse IgG and Alexa Fluor488 goat anti-rabbit (Invitrogen, Carlsbad, CA) for 2 h. The slides were analyzed with an Olympus BX51TF fluorescence microscope (Olympus Corporation, Tokyo, Japan).

Tubular epithelial cellular apoptosis was examined by the TUNEL assay using TdTApopTag ® In Situ Apoptosis Detection Kit (S7165), Merck-Millipore (Merck-Millipore, USA) according to manufacturer instructions. Apoptosis (TUNEL assay) was compared between normoxia control and 7 day hypoxia group. The slides were counterstained with DAPI and mounted with mounting medium containing anti fade. Red colour TUNEL-positive nuclei were identified by using an Olympus BX51TF fluorescence microscope, Japan at 40X magnification equipped with a digital camera. For quantification, the number of positively red stained nuclei per field was determined and an average of ten randomly selected fields per animal was used for analysis. Results have been represented in terms of % positive nuclei.