Secondary alexa fluor 555 conjugated donkey anti rabbit antibody

The samples were taken out from -40°C and cut into 8 μm sections with Cryostat microtome (Thermo Scientific, HM525 NX). The sections were permeabilized with 0.25% Triton X-100 in PBS for 15 min. Then, 5% BSA in PBST buffer was used to block the sections for 1 h. After that, the sections were incubated with KIFC1 rabbit polyclonal antibody (1:100) overnight at 4 °C. The negative control sections were incubated in 5% BSA without primary antibody. They were washed in PBST for 45 min (3 times, 15 min/time). Subsequently, the sections were incubated with Secondary Alexa Fluor 555-conjugated donkey-anti-rabbit antibody (1:500, Beyotime) and anti-α-Tubulin-FITC mouse monoclonal antibody (1:100, Sigma) at room temperature for 1 h. The negative control sections were incubated with just Secondary Alexa Fluor 555-conjugated donkey-anti-rabbit antibody (1:500, Beyotime). After washed 3 times like before, the sections were incubated with DAPI (Beyotime) for 5 min to stain the nucleus. Finally, the sections were mounted with Antifade Mounting Medium (Beyotime) and observed immediately with a confocal laser scanning microscope (Carl Zeiss, CLSM 710).