(22– 24) For ECT2 staining, 1000 tumor cells were evaluated for the most intense nuclear staining (hot spot) according to the method of Iyoda et al. (25) with minor modifications.
Ki-67 staining in tumor cells was evaluated as the percentage of positive tumor cells showing the most intense nuclear staining (hot spot).
Tumor cells showing VEGFsc-152 staining in the cytoplasm was scored according to the intensity of the staining as weak, moderate or intense depending on the area within the tumor that revealed the most intense staining (hot spot).
For HIF-1, similar calculations were done in hot spot areas showing nuclear staining.
After washing, staining was evaluated in five hot spot fields at 400X.
Ki67 was evaluated by the percentage of neoplastic cells showing nuclear staining in the most proliferative area ("hot spot").
Stained epithelial/stromal cell nuclei in these hot spot regions were counted using 40 × objective [ 24].
A mean value of intensity was assigned for specimens in which the staining intensity varied from field to field, and/or from hot spot to hot spot.
Prior to spot picking the gels were hot Coomassie stained according to the method of Reiner Westermeier and Tom Naven [39].
The Virgin Mary has manifested in a dribble of chocolate in a factory and Jesus has turned up in a hot chocolate stain in Colombia.
