Tritc labeled igg

Approximately half of the left kidney of each mouse was subjected to immunofluorescence analysis. Frozen kidney sections were rinsed in phosphate buffered saline (PBS) at room temperature, blocked in 10% control rabbit serum and incubated with TNF-α primary antibody (1:200); sc-52746; Santa Cruz Inc., Dallas, TX, USA). TRITC-labeled IgG (Santa Cruz Inc., USA) was used as a secondary antibody, then the samples were washed three times in PBS, mounted and the fluorescent signals were detected using an Olympus Fluoview FV500 Laser Scanning Confocal System (Tokyo, Japan).

Ovaries from recipient and control mice were fixed with the Tissue-Tek OCT Compound (Sakura Finetek Middle East, Dubai, United Arab Emirates) and sliced in 5 µm thick sections. Slides were washed twice with PBS and kept in blocking solution for 30 min at room temperature. Slides were then incubated with rabbit polyclonal anti-RFP (1∶200; Abcam) or anti-AMH (1∶200, AbD SeroTec) at 4°C overnight. Non-immune immunoglobulins of the same isotype as the primary antibody were used as negative controls. Cells or sections were washed three times with 1×PBS and probed with TRITC–labeled IgG (1∶200, Santa Cruz, CA, U.S.) or FITC-labeled IgG (1∶200, Santa Cruz, CA, U.S.). Fluorescence images were taken using a Leica DMI3000 microscope (Wetzlar, Germany).