Aec substrate 3 amino 9 ethylcarbazole

Frozen sections (6 μm) were immunostained with antirat antibody specific for CD68, a marker for macrophages (OX-38, BD Pharmingen, San Diego, California, USA). Vectastain Elite ABC-peroxidase kit (Vector Laboratories, Burlingame, California, USA) and AEC substrate 3-amino-9-ethylcarbazole (Invitrogen, Grand Island, New York, USA) were used to detect positive immunoreactivity. Negative controls were processed in a similar fashion except that they were not incubated with the primary antibody. Positive cells (reddish brown) were counted in 40× high-power fields in each section and expressed as number of CD68-positive cells per mm².

LV was cut into three slices and snapfrozen in liquid nitrogen-precooled isopentane and stored at −80°C. Frozen sections (6 μm) were immunostained with anti-rat CD4⁺ (a marker for T lymphocytes; OX-38, BD Pharmingen) or anti-rat CD8⁺ antibodies (a marker for cytoxic lymphocytes; OX-8, AbD Serotec) or anti-rat CD68 (a marker for macrophages; OX-38, BD Pharmingen). A Vectastain Elite ABC-peroxidase kit (Vector Laboratories) and the AEC substrate 3-amino-9-ethylcarbazole (Invitrogen) were used to detect positive immunoreactivity. Negative controls were processed in a similar fashion except that they were not incubated with the primary antibody. Positive cells (stained reddish brown) were counted in 40× high-power fields in each section and expressed as number of cells per mm².