Chondrocyte Proliferation Protocol
Imaging & PCNA Staining
Neonatal bone sections are common tissues to study chondrocyte proliferation. Immunohistochemical PCNA labeling in paraffin sections is fairly straight-forward. BIOQUANT’s thresholding tools automatically identify PCNA+ cells based on stain color. Ideally, it's best to keep the counter-stain light to avoid interference with the DAB chromagen.
Digital pathology imaging
Scanning can be done using the BIOQUANT OSTEOIMAGER digital pathology scanner, one of the BIOQUANT digital pathology upgrade kits for you lab’s microscope, or a shared digital pathology core facility.
Proliferation Zone Boundary
Irregular region of interest
Define the contours of the proliferation zone using BIOQUANT’s Irregular Region of Interest tool. BIOQUANT ensures that only chondrocytes within the proliferation zone are counted, and records the area data needed to normalize the number of proliferating cells by the size of the zone.
Detecting PCNA+ Cells
Detection is based on chromagen color and cell morphology. BIOQUANT's Measure Cells tool identifies PCNA+ chondrocytes using the DAB chromagen. Then, labeled cells are passed through size and shape filters to exclude non-specific staining.
BIOQUANT’s Measure Cells tool also filters out cells that have already been counted as we profiles that intersect the edge of the proliferation zone.
Detecting PCNA- Cells
Then Measure Cells identifies PCNA- chondrocytes in the same field of view. These cells are also passed through size and shape filters. Additionally, BIOQUANT knows that PCNA- cells are never PCNA+. Finally, BIOQUANT will not count cells partially in the field of view or that have been previously counted.
auotmatic roi & batch Scripts
Using the Automatic ROI tool and a batch script, BIOQUANT can sequence through every field of view to count cells and still ensure that only cells within the proliferation boundary are counted.
BIOQUANT automatically computes:
- Pr.Zn.Ar (Proliferation Zone Area)
- PCNA+ Ch.N (PCNA+ Chondrocyte Number)
- PCNA- Ch.N (PCNA- Chondrocyte Number)
- Tt.Ch.N (Total Chondrocyte Number)
- PI (Proliferation Index)
- PCNA+ Ch.N / Pr.Z.Ar