Sallee C J, Russell D F
Department of Anesthesiology, Washington University School of Medicine, St. Louis, Missouri 63110.
Biotech Histochem. 1993 Nov;68(6):360-8. doi: 10.3109/10520299309105642.
Agarose was used to embed the brain or spinal cord of lampreys or rats before cutting vibratome sections. Agarose embedding was compatible with immunocytochemistry or the use of horseradish peroxidase as a neuroanatomical tracer. Concentrated agarose with high intrinsic gel strength was optimal for embedding glutaraldehyde fixed neural tissue. A quick procedure was to blot tissue and embed in 5% (w/v) Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in 50 mM neutral-pH TRIS buffer before cutting 50-100 microns vibratome sections. An alternative procedure that improved retention of tissue sections in the agarose was to rinse the tissue in H2O, blot and embed in 5% (w/v) Sigma type I-A or Litex type LSL agarose at 45-55 C dissolved in H2O, then equilibrate the block overnight in buffer. Phosphate buffer prevented complete dissolving of agarose. Tissue could be covalently linked to the embedding matrix using a novel aldehyde-derived agarose (NuFix, FMC BioProducts). Slices of spinal cord from neonatal rats could be cut after embedding in 5% FMC SeaPrep agarose in rat Ringer's at 23-26 C.
在切割振动切片机切片之前，使用琼脂糖包埋七鳃鳗或大鼠的脑或脊髓。琼脂糖包埋与免疫细胞化学或使用辣根过氧化物酶作为神经解剖学示踪剂兼容。具有高固有凝胶强度的浓缩琼脂糖最适合包埋戊二醛固定的神经组织。一种快速方法是吸干组织，然后在45-55℃下将其包埋在溶解于50 mM中性pH Tris缓冲液中的5%（w/v）Sigma I-A型或Litex LSL型琼脂糖中，之后切割50-100微米的振动切片机切片。另一种能提高组织切片在琼脂糖中保留率的方法是将组织在水中冲洗，吸干并包埋在45-55℃下溶解于水中的5%（w/v）Sigma I-A型或Litex LSL型琼脂糖中，然后将包埋块在缓冲液中平衡过夜。磷酸盐缓冲液可防止琼脂糖完全溶解。使用新型醛衍生琼脂糖（NuFix，FMC生物制品公司）可使组织与包埋基质共价连接。新生大鼠的脊髓切片在23-26℃下包埋于含大鼠林格氏液的5% FMC SeaPrep琼脂糖中后即可进行切割。
