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        (求教)再次请问有关冰冻切片的问题

        丁香园论坛

        3077
        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!!!
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