原位聚合酶链式反应（in situ PCR）和原位反转录
<font> <strong>（一）、仪器设备<br />
</strong> <span>英国</span> <span>Thermo Hybaid</span> <span>原位</span> <span>PCR</span> <span>仪。<br />
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</span> <span><span> </span> </span> <span><strong>（二）、操作流程</strong> </span><br />
<span> <span> </span> <strong>1</strong> </span> <strong><span>、原位</span> <span>PCR</span> <span>步骤</span> </strong><br />
<span> </span> <span>1</span> <span>）预处理：<br />
</span> <span> （</span> <span>1</span> <span>）切片常规脱蜡；<br />
</span> <span>（</span> <span>2</span> <span>）</span> <span>0.2mol/L HCl</span> <span>处理</span> <span>10min</span> <span>；<br />
</span> <span> （</span> <span>3</span> <span>）</span> <span>5</span> <span>μ</span> <span>g/ml</span> <span><u>蛋白</u> 酶</span> <span>K</span> <span>消化组织</span> <span>37</span> <span>℃</span> <span>10min</span> <span>；<br />
（</span> <span>4</span> <span>）</span> <span>Nase</span> <span>消化组织</span> <span>37</span> <span>℃</span> <span> 30min</span> <span>；<br />
</span> <span> （</span> <span>5</span> <span>）梯度酒精脱水，室温干燥。<br />
</span> <span>2</span> <span>）原位扩增：<br />
</span> <span> （</span> <span>1</span> <span>）切片滴加特异性序列引物</span> <span>30</span> <span>μ</span> <span>LPCR</span> <span>扩增反应液，覆盖硅化盖玻片，石蜡油封边；<br />
</span> <span>（</span> <span>2</span> <span>）</span> <span>PCR</span> <span>热循环：</span> <span>94</span> <span>℃，</span> <span>1min</span> <span>；</span> <span>55</span> <span>℃，</span> <span>1min</span> <span>；</span> <span>72</span> <span>℃，</span> <span>1.5min</span> <span>，共</span> <span>25</span> <span>～</span> <span>30</span> <span>个循环，</span> <span>72</span> <span>℃延伸</span> <span>10min</span> <span>；<br />
</span> <span>（</span> <span>3</span> <span>）氯仿洗去盖玻片，</span> <span>4</span> <span>％多聚甲醛后固定</span> <span>10min</span> <span>，梯度酒精脱水，干燥。<br />
</span> <span>3) </span> <span>原位杂交：<br />
</span> <span>（</span> <span>1</span> <span>）加地高辛标记探针的杂交液，</span> <span>98</span> <span>℃变性</span> <span>10min</span> <span>，</span> <span>-20</span> <span>℃退火</span> <span>5min</span> <span>，</span> <span>42</span> <span>℃杂交过夜。<br />
</span> <span>（</span> <span>2</span> <span>）杂交后用</span> <span>2×SSC</span> <span>洗涤</span> <span>10min</span> <span>，</span> <span>3</span> <span>次，</span> <span>1×SSC</span> <span>洗涤</span> <span>10min</span> <span>，</span> <span>3</span> <span>次；<br />
</span> <span>（</span> <span>3</span> <span>）缓冲液洗涤</span> <span>10min</span> <span>，</span> <span>3</span> <span>次；<br />
</span> <span>（</span> <span>4</span> <span>）加碱性磷酸酶标记的羊抗地高辛抗体复合物，</span> <span>37</span> <span>℃，</span> <span>2h</span> <span>；<br />
</span> <span>（</span> <span>5</span> <span>）缓冲液洗涤</span> <span>5min</span> <span>，</span> <span>3</span> <span>次；<br />
</span> <span>（</span> <span>6</span> <span>）</span> <span>NBT</span> <span>、</span> <span>BCIP</span> <span>暗处显色，镜下控制，终止显色。<br />
（</span> <span>7</span> <span>）常规脱水：透明、封固。</span><br />
<span><span> </span> <strong>2</strong> </span> <span><strong>、原位</strong> </span> <span><strong>RT-PCR</strong> </span> <span><strong>步骤<br />
</strong> </span> <span>1</span> <span>）预处理：<br />
</span> <span>（</span> <span>1</span> <span>）<u>蛋白</u> 酶</span> <span>K(0.3mg/mL) 54</span> <span>℃消化</span> <span>20min</span> <span>，蒸馏水洗；<br />
</span> <span>（</span> <span>2</span> <span>）</span> <span>95</span> <span>℃加热</span> <span>3min</span> <span>，灭活残存的<u>蛋白</u> 酶。<br />
</span> <span>2</span> <span>）原位扩增：<br />
</span> <span>（</span> <span>1</span> <span>）在有</span> <span>RNA</span> <span>酶抑制剂存在的条件下，用随机六聚物进行逆转录反应；<br />
</span> <span>（</span> <span>2</span> <span>）用热启动法进行</span> <span>PCR</span> <span>扩增。标本加热至</span> <span>75</span> <span>℃时加上反应液，覆以盖玻片，四周用指甲油密封。然后将温度升至</span> <span>95</span> <span>℃，</span> <span>2min</span> <span>。再将热循环仪设定为</span> <span>95</span> <span>℃，</span> <span>45s</span> <span>，</span> <span>55</span> <span>℃，</span> <span>1min</span> <span>和</span> <span>75</span> <span>℃，</span> <span>45s</span> <span>，共</span> <span>26</span> <span>次循环；<br />
</span> <span>（</span> <span>3</span> <span>）扩增结束后，在</span> <span>80</span> <span>℃烤</span> <span>15min</span> <span>～</span> <span>30min</span> <span>。<br />
</span> <span>3</span> <span>）原位杂交：<br />
</span> <span>（</span> <span>1</span> <span>）杂交前标本</span> <span>95</span> <span>℃加热</span> <span>3min</span> <span>；<br />
</span> <span>（</span> <span>2</span> <span>）加上杂交液，湿盒内</span> <span>50</span> <span>℃过夜；</span> <span>杂交液组成：</span> <span>25</span> <span>％硫酸葡聚糖，</span> <span>2×SSC</span> <span>，</span> <span>50</span> <span>％甲酰胺，</span> <span>0.33mg/ml</span> <span>变性的鲑鱼精子</span> <span>DNA</span> <span>，</span> <span>每</span> <span>0</span> <span>．</span> <span>5mL</span> <span>杂交液内含生物素标记探针</span> <span>1ng</span> <span>。<br />
</span> <span>（</span> <span>3</span> <span>）扩增的β</span> <span>-</span> <span>肌动<u>蛋白</u> 和</span> <span>IL-6</span> <span>用</span> <span>DAKO</span> <span>检测试剂盒</span> <span>K600(</span> <span>链霉卵白素，生物素标记的碱性磷酸酶和硝基四氮唑蓝</span> <span>)</span> <span>检测。阳性反应呈紫色。</span> <span><span> </span> </span> </font>
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