Dual enzyme block solution

Sections of 100-μm thickness were prepared from formalin-fixed and paraffin-embedded brain sections from control and from subcortical white matter containing known lesions in patients with MS. They were deparaffinized using standard protocols. Antigen retrieval was performed using sodium citrate buffer (10 mM sodium citrate, 0.05% (v/v) Tween 20). Endogenous peroxidase was inhibited using a dual enzyme block solution (Dako, Carpinteria, CA) and native protein block was achieved with a serum-free protein block solution (Dako). Following a TBS-Tris wash, sections were immunolabeled for 1 h at room temperature using mouse monoclonal anti-PAR1 (1:500, Abcam, San Francisco, CA) or rabbit polyclonal anti-IL1 receptor (1:100, Abcam) as primary antibodies. After a wash with TBS-Tris, sections were incubated with a Powervision poly-HRP anti-rabbit or anti-mouse secondary antibody solution (Dako). Development for visualization of antibody binding within the tissue was achieved using the chromagen 3,3'-diaminobenzidine (Sigma). Hematoxylin (10%; w/v) was used to provide a counterstain. Omissions of the primary or secondary antibodies were used as negative controls.

Cells fixed in 4 % PFA were washed in 1× PBS and incubated for 10′ at RT in a dual enzyme block solution (Dako, Glostrop, Denmark) followed by wash in 1× PBS, and then incubated with protein block solution for 10′ at RT. The cells were then incubated with biotinylated elderberry bark lectin (1:800) (Vector Labs, Burlingame, CA) for 30 min at RT. Following a wash in 1× PBS, the cells were incubated with vectastain ABC-AP (Vector Labs) complex for 30 min at RT. The ABC-AP complex was prepared fresh according to the instructions provided with the vectastain ABC-AP kit. The cells were washed in 1× PBS to remove the ABC-AP complex and incubated with fast red chromagen staining solution (Dako) for 10′ at RT. The cells were rinsed in water and incubated with gill free hematoxylin for 5′. The slides were rinsed in H₂O followed by a gentle wash with 0.5 % ammonium hydroxide. The slides were air dried and mounted on cover slips and the images were captured in tiff format using fluorescence microscope (Nikon) and analyzed.