The Effects of Storage Conditions on the Preservation of Enzymatic Activity in Bone

Abstract

Alkaline phosphatase and acid phosphatase are two major enzymatic measures of osteoblastic and osteoclastic activity, respectively. As a result, the preservation of the enzymes in bone specimens to near in vivo accuracy is essential. Despite standardization of the staining process, several factors related to the storage of blocks and slides before sectioning and staining impact the level of enzymes detected in the tissue. Block condition (intact, faced, or unstained) as well as environment (temperature and length of time in storage) affect alkaline phosphatase preservation while the acid phosphatase enzyme remains unaffected. We conclude that to optimally preserve alkaline phosphatase enzyme, methacrylate-embedded undecalcified murine bones should be stored as intact blocks. After sectioning, the faced blocks should be stored at 4°C for optimal enzyme staining of future sections. Furthermore, it is best to stain sections immediately after sectioning.

Figure 1

Flow diagram of storage conditions. Letters refer to the photographs of stained sections found in Figure 2.

Figure 2

Alkaline phosphatase stain. (A) Section taken from an unfaced block after 1 month storage at 25°C. (B) Section taken from a faced block at the end of 1 month storage at 25°C. (C) Section taken from a faced block at the beginning of 1 month, before storage at 25°C. (D) Section stored on slide, unstained at 25°C for 1 month. (E) Section from unfaced block after 1 month storage at 4°C. (F) Section from a faced block after 1 month of storage at 4°C. (G) Section from a faced block before storage at 4°C. (H) Section stored on slide, unstained at 4°C for 1 month. Original magnification for all micrographs, ×200.