Fong E K, Frank J D, Cartwright M E, Loper A E
Department of Safety Assessment, West Point, PA 19486, USA.
Exp Dermatol. 1996 Feb;5(1):45-8. doi: 10.1111/j.1600-0625.1996.tb00092.x.
A method for preparing skin biopsies for cryosectioning was developed to accurately obtain samples from specific areas of the dermis, while minimizing contamination with epidermal tissue. Routine preparation of 6mm punch biopsies from freshly excised, full-thickness skin produced contraction and folding of the edges of the biopsy prior to mounting for snap-freezing and cryosectioning. Sample orientation was ruined, and cryosections were heterogeneous with respect to dermal structures and/or to dermal and epidermal layers. Biopsy artifacts were prevented by prefreezing skin over dry ice prior to taking biopsies. The biopsies were held frozen on dry ice until they were mounted on cryostat pegs with flattened, frozen OCT surfaces; then they were snap-frozen in chilled OCT in an isopentane bath cooled with liquid nitrogen. The method for determining skin level homogeneity of cryosections consisted of taking 10 mu m cryosections for histology between sections sampled for drug level analysis. The histological sections were fixed in 5% acetic acid in methanol and stained with hematoxylin and eosin to define the skin layers and structures associated with each sample for analysis. Histological sections from prefrozen skin had fewer processing artifacts, and dermal cryosections free of epidermal contamination were dramatically increased compared to the routine procedure.
开发了一种用于制备皮肤活检组织以进行冷冻切片的方法，以便从真皮的特定区域准确获取样本，同时尽量减少表皮组织的污染。从新鲜切除的全层皮肤常规制备6mm打孔活检组织，在固定用于速冻和冷冻切片之前，活检组织边缘会出现收缩和折叠。样本方向被破坏，冷冻切片在真皮结构和/或真皮和表皮层方面是异质的。在活检前通过在干冰上预冻皮肤来防止活检伪像。活检组织在干冰上保持冷冻状态，直到将其安装在冷冻切片机的固定架上，固定架表面为平整的冷冻OCT；然后将它们在液氮冷却的异戊烷浴中的冷冻OCT中速冻。确定冷冻切片皮肤水平均匀性的方法包括在为药物水平分析取样的切片之间取10μm的冷冻切片用于组织学检查。组织学切片用甲醇中的5%乙酸固定，并用苏木精和伊红染色，以确定与每个样本相关的皮肤层和结构用于分析。与常规程序相比，预冻皮肤的组织学切片处理伪像更少，且不含表皮污染的真皮冷冻切片显著增加。
