Second antibody and subsequent incubations were done according to the specifications of a Vectastain ABC Kit (Vector Laboratories) and colour was visualized with 3,3'-diaminobenzidine.
Immunohistochemical stainings were performed using Vector ABC kits (Vector Laboratories) following the manufacturer's recommendations and as described [ 25].
Other reagents, including the secondary antibody, detection kits, and chromogen kits, were purchased from Vector Laboratories, Inc (Burlingame, CA).
Immunohistochemical staining was performed using an indirect immunoperoxidase detection protocol (Vectastain Elite kit; Vector Laboratories) following treatment of sections with hyaluronidase to improve antibody penetration.
Anti-human spectrin antibodies (NCL-SPEC1) were obtained from Vector Laboratories.
Antigen-antibody complexes were detected by a DABNi detection kit according to the supplier's manual (Vector Laboratories, Inc., Burlingame, CA), giving the substrate dark blue color.
The samples were washed extensively in PBS and then incubated with biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA).
DNA was counterstained with Vectashield (Vector Laboratories) containing 1-5 μg/ml 4,6-diamidino-2-phenylindole (DAPI).
"Photoprobe biotin", Vector Laboratories, Inc., www.vectorlabs.com.
Diaminobenzidine (DAB substrate kit for peroxidase, Vector Laboratories, Burlingame, CA) was used as the final chromogen.