Influence of slide coating on tissue shrinkage and counting-frame based analysis in rat spinal cord sections

Quantitative immunohistochemistry is widely used in neuroscience; however, tissue processing-induced shrinkage is often underreported and can bias morphometric analysis. This study examines the effect of slide coating on tissue shrinkage and its impact on ImageJ-based quantification of spinal cord sections. Sections were processed using standard immunohistochemical protocols and mounted on gelatin-coated or uncoated slides. Surface area was measured using ImageJ and expressed in mm². Cell density was determined by counting immunopositive cells within randomly selected non-overlapping counting grids (100 × 100 μm), with ten grids analysed per section and ten sections per group, and expressed as cells/mm². Section thickness was estimated using the focusing depth method and reported in micrometres. Sections mounted on uncoated slides exhibited greater shrinkage along the x-y plane, resulting in reduced surface area and artificially increased cell density estimates when fixed-size counting grids were applied. In contrast, sections mounted on gelatin-coated slides demonstrated reduced section thickness, suggesting preferential shrinkage along the z-axis. These findings demonstrate that minor methodological differences can substantially influence morphometric outcomes. Careful documentation of tissue processing parameters, particularly slide coating, is essential for reliable and reproducible quantitative analyses in neuroscience and pain research.