Histostain plus aec kit

Immunohistochemical analysis to detect MIS RII expression was performed using the Invitrogen Histostain Plus AEC kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Cultured AN3CA cells were harvested and 200 μl of a 1×10⁵ cell/ml suspension was centrifuged with Cytospin (Thermo Electron Corp., Cheshire, UK) at 1000 rpm for 5 min and attached to Probe-on-plus-slides. These slides then were treated with 3% H₂O₂ for 5 min to eliminate endogenous peroxidase activity and washed three times with Tris-buffered saline with 0.1% Tween-20 (T-TBS). After treatment with donkey serum (Invitrogen) for 30 min to block non-specific protein binding, the slides were incubated with rabbit polyclonal anti-human MIS type II receptor antiserum (Massachusetts General Hospital, Boston, MA, USA) (11 (link)) at 4°C overnight. The slides were rinsed in T-TBS three times, and incubated with biotinylated anti-rabbit IgG (Invitrogen) for 30 min. After another three T-TBS rinses, the streptavidin HRP detection system (Invitrogen) was applied to the slides for 30 min to induce the biotin-avidin binding reaction. The slides were treated with 3-amino-9-ethylcarbazole (AEC, Invitrogen) for 10 min at room temperature, counterstained with hematoxylin, then mounted with glycerol gel.

MIS/AMHRII expression was immunocytochemically detected using the Invitrogen Histostain Plus AEC kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Cultured human endometrial cancer cells were harvested, 200 µl of a 1×10⁵ cells/ml suspension was centrifuged with a cytospin (Thermo Electron Corp., Cheshire, WA7, UK) at 1,000 rpm for 5 min, and the cell pellet was transferred to ProbeOn Plus slides (Fisher Scientific). These slides were then treated with 3% hydrogen peroxide for 5 min to eliminate endogenous peroxidase activity and washed three times with PBS. After treatment with donkey serum (Invitrogen) for 30 min to block non-specific protein binding, the slides were incubated with rabbit polyclonal anti-human MIS/AMHRII antiserum (153P) at 4°C overnight. The slides were rinsed in PBS three times and incubated with biotinylated anti-rabbit IgG (Invitrogen) for 30 min. After another three PBS rinses, the streptavidin HRP detection system (Invitrogen) was applied to the slides for 30 min to induce the biotin-avidin binding reaction. The slides were treated with AEC for 10 min at room temperature, counterstained with hematoxylin, and mounted with glycerol gel. Three hundred endometrial cancer cells were counted in five different microscopic fields, and the mean percentage of immunopositive cells was calculated.