Alexa fluor 488 fluoro nanogold

Correlative light and electron microscopy (CLEM) was performed as previously described^{26 (link)}. The liver tissues from PHx Prom1^f/f and Prom1^LKO male mice were fixed with 4% paraformaldehyde, cryoprotected with 2.3 M sucrose (0.1 M phosphate buffer) and frozen in liquid nitrogen. The frozen tissues were cut to 1-um-thick at −100°C with Leica EM UC7 ultramicrotome. The sections were labeled at 4°C overnight using an anti-PROM1 rat monoclonal antibody (13A4, 1:200) and visualized using an Alexa Fluor 488-Fluoro Nanogold (Nanoprobes, 1:100). Cover slipped sections were detected with a confocal microscope (Zeiss, LSM700) with a differential interference contrast setting to find specific areas for later examination by electron microscopy. After coverslips had been floated off, silver enhancement was performed using HQ silver enhancement kit (Nanoprobes). After prepared for electron microscopy, areas of interest were excised and cut into 70–90 nm thick. The samples were observed in an electron microscope (JEM 1010; JEOL, Tokyo, Japan).

First, the sections were removed from the PBS and placed in a fresh solution of 1% sodium borohydride in 0.1 M PBS for 30 minutes at room temperature (RT). They were then incubated for 30 minutes at RT in 1% bovine serum albumin and 0.1% gelatin in PBS at pH 7.4 (PBS-BSA). Next, sections were incubated for 48 hours with the following primary antibodies in blocking solution: rabbit anti-p-Syn I antibody (ser-62/67 and ser-553; 1∶1000 dilution; Santa Cruz Biotechnology), anti-VGLUT1 antibody (BNPI; 1∶1000 dilution; Santa Cruz Biotechnology), and anti-VGAT antibody (1∶1000 dilution; Merck Millipore). Subsequently, the sections were incubated with secondary antibodies (Alexa Fluor488 FluoroNanogold,Nanoprobes, NY, USA) diluted 1∶100 in PBS-BSA with 1% goat serum for 1 hour at RT, and then they were post-fixed with 1% glutaraldehyde in PBS for 10 minutes. Finally, silver enhancement with a HQ silver EM intensification kit (Nanoprobes) was performed for 6 minutes and then the sections were rinsed three times in deionized water for 10 min.