IMPORTANCE OF FIXATION IN IMMUNOHISTOCHEMISTRY - USE OF FORMALDEHYDE SOLUTIONS AT VARIABLE PH FOR THE LOCALIZATION OF TYROSINE-HYDROXYLASE
IMPORTANCE OF FIXATION IN IMMUNOHISTOCHEMISTRY - USE OF FORMALDEHYDE SOLUTIONS AT VARIABLE PH FOR THE LOCALIZATION OF TYROSINE-HYDROXYLASE
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DOI：
10.1177/29.7.6167611
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发表时间：
1981-01-01
影响因子：
3.2
通讯作者：
PUJOL, JF
中科院分区：
文献类型：
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作者：
BEROD, A;HARTMAN, BK;PUJOL, JF
在免疫组织化学中，足够的固定剂不仅需要快速和完全固定抗原，而且还需要充分保存其免疫反应性并维持其对免疫化学试剂的可及性以进行定位。特定抗原的最佳固定条件需要在这些相反的变量之间进行折衷，并且可以通过制备一系列固定程度逐渐增加的组织来确定。如果使用这样一个系列的定位是不可用的，结果将是足够的，但不是最佳的。在本研究中，这一原则是证明使用的酪氨酸羟化酶在多巴胺能系统与甲醛作为固定剂的本地化。甲醛固定的速率和程度高度依赖于pH值。通过在pH 6.5下用甲醛灌注组织[大鼠脑]（其中固定速率极慢），可以将固定剂快速均匀地分布到组织中。通过突然改变为更高pH的甲醛灌注液，交联反应迅速增加。该2步固定程序提供了获得抗原的快速和均匀固定的方法，从而可以避免其移位。最终固定程度由第二次固定液的持续时间和pH值控制。通过增加第二溶液的pH值获得的结果表明，使用非常碱性的甲醛溶液（pH 11）可以获得多巴胺能系统中的酪氨酸羟化酶与甲醛的完全固定，同时仍然保持酶的免疫反应性。在较低pH下实现的定位似乎是足够的，直到将其与第二步中通过在pH 11下灌注获得的结果进行比较。
Adequate fixative in immunohistochemistry requires not only a rapid and total immobilization of the antigen, but also a sufficient preservation of its immunoreactivity and maintenance of its accessibility to the immunochemical reagents for localization. The optimal fixation condition for a specific antigen necessitates a compromise between these opposing variables and can be determined by the preparation of a series of tissues with a progressively increasing degree of fixation. If localization using such a series is not available the results will be adequate but less than optimal. In the present study, this principle is demonstrated using the localization of tyrosine hydroxylase in the dopaminergic system with formaldehyde as the fixative. The rate and degree of fixation with formaldehyde was highly pH dependent. By perfusing the tissue [rat brain] with formaldehyde at pH 6.5 (where the rate of fixation is extremely slow) it is possible to rapidly distribute the fixative homogeneously into the tissue. By suddenly changing to a formaldehyde perfusate of higher pH, the cross-linking reaction is rapidly increased. This 2-step fixation procedure provides a means of obtaining a rapid and uniform immobilization of the antigen, so that its translocation can be avoided. The final degree of fixation is controlled by the duration and pH of the 2nd fixative solution. The results obtained by increasing the pH of the 2nd solution demonstrated that complete fixation of tyrosine hydroxylase in the dopaminergic system with formaldehyde may be obtained using a very basic formaldehyde solution (pH 11) while still retaining immunoreactivity of the enzyme. The localization that was achieved at lower pH appeared adequate until it was compared to the results obtained by perfusion at pH 11 in the 2nd step.