EMBEDDING OF NEURAL TISSUE IN AGAROSE OR GLYOXYL AGAROSE FOR VIBRATOME SECTIONING
EMBEDDING OF NEURAL TISSUE IN AGAROSE OR GLYOXYL AGAROSE FOR VIBRATOME SECTIONING
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DOI：
10.3109/10520299309105642
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发表时间：
1993-11-01
影响因子：
1.6
通讯作者：
RUSSELL, DF
中科院分区：
文献类型：
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作者：
SALLEE, CJ;RUSSELL, DF
用琼脂糖包埋七鳃鳗或大鼠的脑或脊髓，然后切开振动切片。琼脂糖包埋与免疫细胞化学或辣根过氧化物酶作为神经解剖示踪剂的使用是兼容的。具有较高固有凝胶强度的浓缩琼脂糖包埋戊二醛固定的神经组织是最佳的。一种快速的方法是将组织印迹并包埋在5%(w/v)的Sigma I-A型或Litex型LSL琼脂糖凝胶中，在45-55℃下溶解在50 mm中性pH Tris缓冲液中，然后切割50-100微米的振荡器切片。提高组织切片在琼脂糖中保留率的另一种方法是用水冲洗组织，将组织在45-55℃的45-55℃溶解在H2O中，将组织吸出并包埋在5%(w/v)Sigma I-A型或Litex型LSL琼脂糖中，然后在缓冲液中过夜平衡块。磷酸盐缓冲液可防止琼脂糖凝胶完全溶解。利用一种新型的醛衍生琼脂糖凝胶(NuFix(R)，FMC BioProducts)，组织可以共价连接到包埋基质上。新生大鼠脊髓切片在23~26℃下以5%FMC SeaPrep(R)琼脂糖包埋于大鼠Ringer‘s中即可切开。
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 mum 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(R), FMC BioProducts). Slices of spinal cord from neonatal rats could be cut after embedding in 5% FMC SeaPrep(R) agarose in rat Ringer's at 23-26 C.