Alexa568 anti rabbit

Paraffin sections were rehydrated as described before. For antigen retrieval, slides were transferred to a plastic cuvette with citrate buffer (recipe see Schlosser, 2008) and heated in a microwave (Bosch HMT 72M 420) for 10 minutes at 180W. Subsequently, slides were placed at room temperature (RT, 21-25°C) to cool down for at least 5 minutes and then rinsed in distilled water for 1 minute. Afterwards, slides were rinsed with PBS (3 x 5 minutes), placed into a wet chamber and blocked with antibody diluent (DAKO) for 1 hour at RT. Primary antibodies against proliferating cell nuclear antigen (PCNA-1, 1/200, sc-7907, Santa Cruz; PCNA-2, 1/200, M087901, Dako) were applied overnight at 4°C. On the next day, slides were rinsed in PBS (3 x 5 minutes), blocked and incubated with secondary antibodies (Alexa-488 anti-mouse, #A28175, Thermo Fisher Scientific and Alexa-568 anti-rabbit, #A-11011, Thermo Fisher Scientific) for 1 hour at RT. Afterwards, slides were rinsed with PBS (3 x 5 minutes), quickly washed with distilled water and cover-slipped with Fluoroshield with DAPI (Sigma, F6057). Slides were photographed using a Zeiss Axioplan Microscope equipped with a Spot camera and the Zeiss Axioplan software.