ST of the knee was obtained during arthroscopy and fixed in formalin. IHC staining was applied to the sectioned ST. We used anti–A antibody (Ab) (1:100 diluted, ab2521; Abcam, Cambridge, UK), anti–B Ab (1:100 diluted, ab2524; Abcam, Cambridge, UK), anti–H Ab (1:100 diluted, ab3355; Abcam, Cambridge, UK), and anti–LeY Ab (1:100 diluted, ab3359; Abcam, Cambridge, UK) as the primary Ab and biotinylated anti–mouse IgM as the secondary Ab. A Vector ABC system (PV-6002, Zsbio, Beijing, China) containing avidin-HRP (horseradish peroxidase) was used with 3,3′-diaminobenzidine as a substrate. Slides were counterstained with haematoxylin and eosin.
Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative way. The distribution of positive cells was graded as follows: none (not stained) = 0, focal (less than one-third of cells of the same kind) = 1, multifocal (less than two-thirds of cells of the same kind) = 2, and diffuse (more than two-thirds of cells of the same kind) = 3. The intensity of staining was graded as follows: none (not stained) = 0, slight (light yellow) = 1, mild (brown) = 2, and strong (dark brown) = 3. The scores of distribution and intensity were added as the final score [20] (link). In addition, the localisation of staining was recorded.
Both the distribution (the percentage of positive cells) and the intensity of staining were assessed in a semiquantitative way. The distribution of positive cells was graded as follows: none (not stained) = 0, focal (less than one-third of cells of the same kind) = 1, multifocal (less than two-thirds of cells of the same kind) = 2, and diffuse (more than two-thirds of cells of the same kind) = 3. The intensity of staining was graded as follows: none (not stained) = 0, slight (light yellow) = 1, mild (brown) = 2, and strong (dark brown) = 3. The scores of distribution and intensity were added as the final score [20] (link). In addition, the localisation of staining was recorded.