Cells grown in 8-well chamber slides to 70% confluence were fixed with 4% paraformaldehyde (15 min at room temperature (RT)), and permeabilized with 100% ice cold methanol (10 min at 20°C). The slides were incubated with 10% normal goat serum (ab7481) in PBS for 1 hr, and incubated with anti-maspin (1:100) antibody alone or in a combination with either anti-lamin B (1:50), anti-HDAC1 (1:50) or anti-GRP78 (1:50) at 4°C overnight. Cells were washed and incubated for 2 hrs at room temperature (RT) with Alexa Fluor 488 (1:500) alone or in combination with Alexa Fluor 594 (1:500). The nuclei were counterstained with DAPI.
DU145 cells infected with adenovirus expressing either maspinWT or maspinD346E Confocal immunofluorescence imaging of maspin (green) and nuclear envelope marker lamin B (red) in DU145 cells after infection.

Slides were pretreated with Hydrogen Peroxide Block followed by 15% normal goat serum (Abcam, Cambridge, UK) for 1 hour. The primary antibody, anti-PTX3, N-terminal antibody produced in rabbit was diluted 1:300 in PBS with 2.5% goat serum (ab7481), applied to slides and incubated for 3 hours at 4°C. The primary antibody was omitted in the negative controls. The PTX3 binding was revealed using a universal secondary antibody polymer formulation conjugated to horseradish peroxidase (HRP). The HRP activity was subsequently visualized with diaminobenzidine (DAB) substrate/chromogen and counterstaining with hematoxylin was performed.