Slides of paraffin-embedded primary HCC tissues were used for immunochemistry and stained with antibodies against Prp19 and Cdc5L. The exact procedure and the semi-quantitative method were reported elsewhere [27 (link)]. Images were processed with a Nikon microscope with NIS Element F3.2 software (Nikon, Melville, NY, USA).
When subjected to immunofluorescence assay, unfixed frozen sections were generally processed as described below, briefly. First, sections were incubated with rabbit anti-human Prp19 polyclonal antibody (1:250) and mouse anti-human Cdc5L Monoclonal antibody (1:20) overnight at 4 °C after blocked by Phosphate buffered saline (PBS) containing 5% Bovine serum albumin (BSA). They were then washed by PBS carefully. These sections were incubated with Alexa Fluor® 647-conjugated AffiniPure Donkey Anti-Rabbit IgG and Alexa Fluor® 488-conjugated AffiniPure Donkey Anti-Mouse IgG (both 1:500) at room temperature for 2 h (Jackson ImmunoResearch Inc., West Grove, PA, USA). Finally, sections were added with Mounting Medium for Flurescence with DAPI and detected by Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).
When subjected to immunofluorescence assay, unfixed frozen sections were generally processed as described below, briefly. First, sections were incubated with rabbit anti-human Prp19 polyclonal antibody (1:250) and mouse anti-human Cdc5L Monoclonal antibody (1:20) overnight at 4 °C after blocked by Phosphate buffered saline (PBS) containing 5% Bovine serum albumin (BSA). They were then washed by PBS carefully. These sections were incubated with Alexa Fluor® 647-conjugated AffiniPure Donkey Anti-Rabbit IgG and Alexa Fluor® 488-conjugated AffiniPure Donkey Anti-Mouse IgG (both 1:500) at room temperature for 2 h (Jackson ImmunoResearch Inc., West Grove, PA, USA). Finally, sections were added with Mounting Medium for Flurescence with DAPI and detected by Leica TCS SP5 confocal microscope (Leica Microsystems, Wetzlar, Germany).