In chondrogenic medium, positive collagen 2 staining was visible around some of the tendon-derived fibroblasts and the tendon-derived fibroblasts showed upregulation of COL2A1 and COL10A1.
Deconvolution of the hematoxylin channel allowed the generation of a nuclear chromatin density ratio (CDR), defined as the ratio of nuclear area with medium-positive hematoxylin staining intensity (corresponding to "active" nuclei) to nuclear area with strong-positive hematoxylin staining intensity (corresponding to "inactive" nuclei).
The strong, medium and weak expressions in IHC were defined as positive staining and, in contrast, the absent expression as negative staining.
The positive index (PI) was calculated using the following formulation: PI = i × p, where i is intensity of staining (0 for negative, blue; 1 for weakly-positive, light yellow; 2 for medium positive, yellow; 3 for strong positive, brown), and p is positive percentage of staining (1 for ≦10%; 2 for 11%-50 11%-50r 51%-75%; 4 for >75%) [ 11].
These algorithms were used to calculate the average positive intensity (API), as well as the area of positive staining, and the percentage of weak (1+), medium (2+), and strong (3+) positive staining.
In order to determine the correlation of Myc with MCL1, IHC staining of Myc and MCL1 was performed in 142 breast tumor tissue array samples and the staining was graded as weak positive (G I), medium positive (G II) and strong positive (G III).
Negative controls were glioblastoma cells showing mesenchymal phenotype but not exposed to adipogenic medium; positive controls consisted of marrow stromal cells exposed to adipogenic medium and stained with Oil Red-O.
The same settings were used for each stain across all images, and the area of positive staining was calculated by dividing the total number of positive pixels (weak, medium and strong staining) by the total number of pixels in the image (positive + negative pixels) to yield the overall percentage of positive pixels.
PDZK1 expression, as a cytoplasmic marker, was scored on a semi-quantitative scale depending on intensity of cytoplasmic staining: ranging from 0 3, where 0 is negative, 1 is weakly positive, 2 is medium positive and 3 is strongly positive.
After 35 d, Con-P1rAC showed the strongest positive staining among four cultures and for both cells, and collagen II deposition was greater in chondrogenic medium than in growth medium, 4(c), 4 e), and 4(f)).
