(求教)再次请问有关冰冻切片的问题
丁香园
2410
我现在准备做大鼠心肌组织的冰冻切片（研究心肌细胞膜上的钾离子通道蛋白），按照丁香园学友的帮忙，取标本后先放在液氮中，随后4％多聚甲醛磷酸缓冲液后固定标本后，再放入20％蔗糖PB液，直至组织块沉底。本打算今天高高兴兴去做冰冻切片免疫组化（SP法），但是帮我做切片的病理科技术员说：蔗糖泡过的标本非常软，均无法切片，以前他也遇到过类似的问题。具体原因他也解答不清。不知哪位学友帮忙解答？
1 是否我们医院病理科冰冻切片机比较不好，无法切出理想的片来？
2 是否与没用OCT包埋有关？（因为我是用普通胶水代替）
3 先冰冻切片随后再多聚甲醛磷酸缓冲液固定标本及20％蔗糖PB处理是否可行？
实在没有办法，郁闷之下再去查找文献，发现国外学者有的是把-80度冰冻后的标本在冰冻切片机上先做切片，然后在4％多聚甲醛中固定20分钟，随后就加入一抗，在文献中未提到用蔗糖。
（Immunohistochemistry： Atrial and ventricular tissues were dissected from five explanted canine hearts and placed in isobutanol precooled to
80°C, which was then cooled further in liquid nitrogen to
ensure rapid and even freezing. Frozen tissue was subsequently
stored at 80°C until cryosectioning, which took
place at 20°C. Serial 14-m sections were cut with a Leica
CM1900 cryostat and left to dry before storage at 80°C.
Before the application of antibodies to the sections, the tissue
was fixed for 20 min with 3%-paraformaldehyde solution.
The primary antibodies (anti-Kir2.1, anti-Kir2.3, or anti-Ncadherin)
were in contact with the sections for 90 min at
room temperature. This was followed by three washes with
PBS and the application of the secondary antibodies (antirabbit
IgG-Cy3 or anti-mouse IgM-Cy5) for 45 min at room
temperature. After being washed, the sections were mounted
using Immuno Floure. 文献：Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle Am J Physiol Heart Circ Physiol 283: H1123–H1133, 2002.）
我的问题是：不知哪位学友有用到类似方法？是否可行？而他的试验结果是通过免疫荧光方法分析，是否他实验的步骤也适合我实验中的SP方法（最后用DAB染色）？
焦急之中，望帮忙，万分感谢！！！
1 是否我们医院病理科冰冻切片机比较不好，无法切出理想的片来？
2 是否与没用OCT包埋有关？（因为我是用普通胶水代替）
3 先冰冻切片随后再多聚甲醛磷酸缓冲液固定标本及20％蔗糖PB处理是否可行？
实在没有办法，郁闷之下再去查找文献，发现国外学者有的是把-80度冰冻后的标本在冰冻切片机上先做切片，然后在4％多聚甲醛中固定20分钟，随后就加入一抗，在文献中未提到用蔗糖。
（Immunohistochemistry： Atrial and ventricular tissues were dissected from five explanted canine hearts and placed in isobutanol precooled to
80°C, which was then cooled further in liquid nitrogen to
ensure rapid and even freezing. Frozen tissue was subsequently
stored at 80°C until cryosectioning, which took
place at 20°C. Serial 14-m sections were cut with a Leica
CM1900 cryostat and left to dry before storage at 80°C.
Before the application of antibodies to the sections, the tissue
was fixed for 20 min with 3%-paraformaldehyde solution.
The primary antibodies (anti-Kir2.1, anti-Kir2.3, or anti-Ncadherin)
were in contact with the sections for 90 min at
room temperature. This was followed by three washes with
PBS and the application of the secondary antibodies (antirabbit
IgG-Cy3 or anti-mouse IgM-Cy5) for 45 min at room
temperature. After being washed, the sections were mounted
using Immuno Floure. 文献：Differential distribution of Kir2.1 and Kir2.3 subunits in canine atrium and ventricle Am J Physiol Heart Circ Physiol 283: H1123–H1133, 2002.）
我的问题是：不知哪位学友有用到类似方法？是否可行？而他的试验结果是通过免疫荧光方法分析，是否他实验的步骤也适合我实验中的SP方法（最后用DAB染色）？
焦急之中，望帮忙，万分感谢！！！
