CN111811909A - 一种组织自发荧光淬灭剂的制备及应用 - Google Patents
一种组织自发荧光淬灭剂的制备及应用 Download PDFInfo
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Abstract
本发明公开了一种组织自发荧光淬灭剂,该荧光淬灭剂包括由以下组分构成:组织自发荧光淬灭剂A和组织自发荧光淬灭剂B:组织自发荧光淬灭剂A为pH7.4的磷酸缓冲液与硼氢化钠的混合;组织自发荧光淬灭剂B为苏丹黑B与无水乙醇的混合液,一种组织自发荧光淬灭剂的应用方法,包括如下步骤构成:S1.石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min、二甲苯Ⅱ15min、无水乙醇Ⅰ5min、无水乙醇Ⅱ5min、85%酒精5min、75%酒精5min、蒸馏水洗,S2.抗原修复:组织切片置于盛满EDTA抗原修复缓冲液即pH8.0的修复盒中,本发明的目的在于提供一种组织自发荧光淬灭剂的制备及应用,该试剂制备简单快捷,原料易得,可以有效降低组织自发荧光提高免疫荧光分析中的信噪比。
Description
技术领域
本发明涉及生物医药应用领域,尤其是涉及一种组织自发荧光淬灭剂的制备及应用。
背景技术
免疫荧光技术是以抗体与抗原特异性结合为基础的一种实验技术,由于操作简单、效率高、特异性强等优势在基础研究和临床实验中被广泛应用。但组织中的自发荧光对组织切片的检测结果造成很大干扰,甚至导致染色失败。组织自发荧光产生的主要原因是来自于组织本身的组分。主要包括丰富的内源性过氧化物酶、胶原蛋白、弹性蛋白、脂褐素等。除去组织切片自身原因,组织固定中经常使用的甲醛、戊二醛和福尔马林等固定液也会造成组织产生自发荧光。
用于减少组织自发荧光的一种方法是光漂白法,该方法需要组织切片长时间暴露于高强度UV辐射下,组织中的成分发生不可逆的光氧化作用。该种方法耗时时间长,且大多数免疫组化实验室不具备该方法所需的仪器设备。
为此,提出一种组织自发荧光淬灭剂的制备及应用。
发明内容
本发明的目的在于提供一种组织自发荧光淬灭剂的制备及应用,该试剂制备简单快捷,原料易得,可以有效降低组织自发荧光提高免疫荧光分析中的信噪比,以解决上述背景技术中提出的问题。
为实现上述目的,本发明提供如下技术方案:
一种组织自发荧光淬灭剂,该荧光淬灭剂包括由以下组分构成:
组织自发荧光淬灭剂A和组织自发荧光淬灭剂B:组织自发荧光淬灭剂A为pH7.4的磷酸缓冲液与硼氢化钠的混合;组织自发荧光淬灭剂B为苏丹黑B与无水乙醇的混合液。
一种组织自发荧光淬灭剂的应用方法,包括如下步骤构成:
S1.石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min、二甲苯Ⅱ15min、无水乙醇Ⅰ5min、无水乙醇Ⅱ5min、85%酒精5min、75%酒精5min、蒸馏水洗;
S2.抗原修复:组织切片置于盛满EDTA抗原修复缓冲液即pH8.0的修复盒中于蒸锅内进行抗原修复,温度达到95℃后计时30min,此过程中应防止缓冲液过度蒸发,切勿干片,自然冷却;
S3.画圈自发荧光淬灭:切片稍甩干后用组化笔在组织周围画圈,防止抗体流走,在圈内加入自发荧光淬灭剂A,室温孵育15min,流水冲洗10min,在圈内加入自发荧光淬灭剂B,室温孵育10min,流水冲洗10min;
S4.血清封闭:在圈内滴加5%BSA室温孵育30min;
S5.加一抗:轻轻甩掉封闭液,在切片上滴加PBS按一定比例配好的一抗,切片平放于湿盒内4℃孵育过夜,湿盒内加少量水防止抗体蒸发;
S6.加二抗:玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加与一抗相应种属的二抗覆盖组织,避光室温孵育1h。
S7.DAPI复染细胞核:玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min;
S8.封片:玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后用抗荧光淬灭封片剂封片;
S9.镜检:显微镜下选取视野拍照。
优选的,在所述S3中孵育完自发荧光淬灭剂B后,组织会被染为蓝黑色,该结果不会影响后续实验结果。
优选的,所述S2中,自然冷却后将玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min。
与现有技术相比,本发明的有益效果是:
本发明的目的在于提供一种组织自发荧光淬灭剂的制备及应用,该试剂制备简单快捷,原料易得,可以有效降低组织自发荧光提高免疫荧光分析中的信噪比。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图是本发明的一些实施方式,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为本发明未使用组织自发荧光淬灭剂结果(肝脏-CD8)图;
图2为本发明使用组织自发荧光淬灭剂结果(肝脏-CD8,同一视野)图;
图3为本发明未使用组织自发荧光淬灭剂结果(肾脏-CD31)图;
图4为本发明使用组织自发荧光淬灭剂结果(肾脏-CD31,同一视野)图;
图5为本发明实施例二中的为使用组织自发荧光淬灭剂小鼠肝脏CD8免疫荧光结果图;
图6为本发明实施例三中的为使用组织自发荧光淬灭剂小鼠肾脏CD31免疫荧光结果图;
图7为本发明实施例四中的使用组织自发荧光淬灭剂大鼠心脏Cx43免疫荧光结果图。
具体实施方式
下面将结和本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
请参阅图1至图7,本发明提供四种实施例:
实施例一:
组织自发荧光淬灭剂A配制:分别称取NaCl 8.0g,KCl 0.2g,Na2HPO4 1.44g,KH2PO4 0.24g,加入超纯水溶解定容1000ml,得到pH为7.4的磷酸缓冲液。称取10mg硼氢化钠溶于10ml pH为7.4的磷酸缓冲液中。
组织自发荧光淬灭剂B配制:称取苏丹黑B 0.5g,于70%酒精中溶解2天,过滤备用。
实施例二:
组织自发荧光淬灭剂使用方法:小鼠肝脏CD8免疫荧光
S1.石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min、二甲苯Ⅱ15min、无水乙醇Ⅰ5min、无水乙醇Ⅱ5min、85%酒精5min、75%酒精5min、蒸馏水洗;
S2.抗原修复:组织切片置于盛满EDTA抗原修复缓冲液即pH8.0的修复盒中于蒸锅内进行抗原修复,温度达到95℃后计时30min,此过程中应防止缓冲液过度蒸发,切勿干片,自然冷却后将玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min;
S3.画圈自发荧光淬灭:切片稍甩干后用组化笔在组织周围画圈,防止抗体流走,在圈内加入自发荧光淬灭剂A,室温孵育15min,流水冲洗10min,在圈内加入自发荧光淬灭剂B,室温孵育10min,流水冲洗10min,孵育完自发荧光淬灭剂B后,组织会被染为蓝黑色,该结果不会影响后续实验结果;
S4.血清封闭:在圈内滴加5%BSA室温孵育30min;
S5.加一抗:轻轻甩掉封闭液,在切片上滴加PBS按一定比例配好的CD8一抗,切片平放于湿盒内4℃孵育过夜,湿盒内加少量水防止抗体蒸发;
S6.加二抗:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加GoatAnti-Rabbit IgG H&L(FITC)二抗覆盖组织,避光室温孵育1h;
S7.DAPI复染细胞核:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min;
S8.封片:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后用抗荧光淬灭封片剂封片;
S9.镜检:显微镜下选取视野拍照。
图5为使用组织自发荧光淬灭剂小鼠肝脏CD8免疫荧光结果,由结果可知,本试剂明显降低组织自发荧光,提高免疫荧光信噪比。
实施例三:
组织自发荧光淬灭剂使用方法:小鼠肾脏CD31免疫荧光
S1.石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min、二甲苯Ⅱ15min、无水乙醇Ⅰ5min、无水乙醇Ⅱ5min、85%酒精5min、75%酒精5min、蒸馏水洗;
S2.抗原修复:组织切片置于盛满EDTA抗原修复缓冲液即pH8.0的修复盒中于蒸锅内进行抗原修复,温度达到95℃后计时30min,此过程中应防止缓冲液过度蒸发,切勿干片,自然冷却后将玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min;
S3.画圈自发荧光淬灭:切片稍甩干后用组化笔在组织周围画圈,防止抗体流走,在圈内加入自发荧光淬灭剂A,室温孵育15min,流水冲洗10min。在圈内加入自发荧光淬灭剂B,室温孵育10min,流水冲洗10min,孵育完自发荧光淬灭剂B后,组织会被染为蓝黑色,该结果不会影响后续实验结果;
S4.血清封闭:在圈内滴加5%BSA室温孵育30min;
S5.加一抗:轻轻甩掉封闭液,在切片上滴加PBS按一定比例配好的CD31一抗,切片平放于湿盒内4℃孵育过夜,湿盒内加少量水防止抗体蒸发;
S6.加二抗:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加GoatAnti-Rabbit IgG H&L(FITC)二抗覆盖组织,避光室温孵育1h;
S7.DAPI复染细胞核:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min;
S8.封片:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后用抗荧光淬灭封片剂封片;
S9.镜检:显微镜下选取视野拍照。
图6为使用组织自发荧光淬灭剂小鼠肾脏CD31免疫荧光结果,由结果可知,本试剂明显降低组织自发荧光,提高免疫荧光信噪比。
实施例四:
组织自发荧光淬灭剂使用方法:大鼠心脏Cx43免疫荧光
S1.石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min、二甲苯Ⅱ15min、无水乙醇Ⅰ5min、无水乙醇Ⅱ5min、85%酒精5min、75%酒精5min、蒸馏水洗;
S2.抗原修复:组织切片置于盛满EDTA抗原修复缓冲液即pH8.0的修复盒中于蒸锅内进行抗原修复,温度达到95℃后计时30min,此过程中应防止缓冲液过度蒸发,切勿干片,自然冷却后将玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min;
S3.画圈自发荧光淬灭:切片稍甩干后用组化笔在组织周围画圈,防止抗体流走,在圈内加入自发荧光淬灭剂A,室温孵育15min,流水冲洗10min。在圈内加入自发荧光淬灭剂B,室温孵育10min,流水冲洗10min,孵育完自发荧光淬灭剂B后,组织会被染为蓝黑色,该结果不会影响后续实验结果;
S4.血清封闭:在圈内滴加5%BSA室温孵育30min;
S5.加一抗:轻轻甩掉封闭液,在切片上滴加PBS按一定比例配好的Cx43一抗,切片平放于湿盒内4℃孵育过夜,湿盒内加少量水防止抗体蒸发;
S6.加二抗:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加GoatAnti-Rabbit IgG H&L(FITC)二抗覆盖组织,避光室温孵育1h;
S7.DAPI复染细胞核:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min;
S8.封片:玻片置于PBS即pH7.4,中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后用抗荧光淬灭封片剂封片;
S9.镜检:显微镜下选取视野拍照。
图7为使用组织自发荧光淬灭剂大鼠心脏Cx43免疫荧光结果,由结果可知,本试剂明显降低组织自发荧光,提高免疫荧光信噪比。
有益效果:
本发明的目的在于提供一种组织自发荧光淬灭剂的制备及应用,该试剂制备简单快捷,原料易得,可以有效降低组织自发荧光提高免疫荧光分析中的信噪比。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Claims (4)
1.一种组织自发荧光淬灭剂,其特征在于:该荧光淬灭剂包括由以下组分构成:
组织自发荧光淬灭剂A和组织自发荧光淬灭剂B:组织自发荧光淬灭剂A为pH7.4的磷酸缓冲液与硼氢化钠的混合;组织自发荧光淬灭剂B为苏丹黑B与无水乙醇的混合液。
2.根据权利要求1所述的一种组织自发荧光淬灭剂的应用方法,其特征在于,包括如下步骤构成:
S1.石蜡切片脱蜡至水:依次将切片放入二甲苯Ⅰ15min、二甲苯Ⅱ15min、无水乙醇Ⅰ5min、无水乙醇Ⅱ5min、85%酒精5min、75%酒精5min、蒸馏水洗;
S2.抗原修复:组织切片置于盛满EDTA抗原修复缓冲液即pH8.0的修复盒中于蒸锅内进行抗原修复,温度达到95℃后计时30min,此过程中应防止缓冲液过度蒸发,切勿干片,自然冷却;
S3.画圈自发荧光淬灭:切片稍甩干后用组化笔在组织周围画圈,防止抗体流走,在圈内加入自发荧光淬灭剂A,室温孵育15min,流水冲洗10min,在圈内加入自发荧光淬灭剂B,室温孵育10min,流水冲洗10min;
S4.血清封闭:在圈内滴加5%BSA室温孵育30min;
S5.加一抗:轻轻甩掉封闭液,在切片上滴加PBS按一定比例配好的一抗,切片平放于湿盒内4℃孵育过夜,湿盒内加少量水防止抗体蒸发;
S6.加二抗:玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后在圈内滴加与一抗相应种属的二抗覆盖组织,避光室温孵育1h;
S7.DAPI复染细胞核:玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min。切片稍甩干后在圈内滴加DAPI染液,避光室温孵育10min;
S8.封片:玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min,切片稍甩干后用抗荧光淬灭封片剂封片;
S9.镜检:显微镜下选取视野拍照。
3.根据权利要求2所述的一种组织自发荧光淬灭剂的应用方法,其特征在于:在所述S3中孵育完自发荧光淬灭剂B后,组织会被染为蓝黑色,该结果不会影响后续实验结果。
4.根据权利要求2所述的一种组织自发荧光淬灭剂的应用方法,其特征在于:所述S2中,自然冷却后将玻片置于PBS即pH7.4中在脱色摇床上晃动洗涤3次,每次5min。
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