IHC Technique Guide: Tips to Prevent Tissue Detachment
Preface:
“Tissue detachment” is something every technician wants to avoid, as it means the work of one or even two days goes down the drain, which is quite frustrating. There are many reasons for tissue detachment in immunohistochemical staining operations, such as poor tissue processing, incomplete dewaxing, high-temperature antigen retrieval, primary antibody incubation, etc. Details determine success or failure. Here are a few simple tips to prevent detachment.
Proper Baking of Slides
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Baking Temperature and Time:Baking below the wax melting temperature can be done overnight, or at 55~65°C, ensuring the moisture is completely dried (1-3 hours). Excessive temperature can lead to antigen loss or weakening. In practice, pay attention to the melting point of the paraffin used and whether the next step will be performed immediately. Slices can be directly frozen for storage and baked in advance before IHC operations.
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Air-dry or Spin-dry Moisture Before BakingIf slides are baked immediately after sectioning, because the wax melts quickly, moisture may accumulate under the tissue due to surface tension, forming water droplets. This can cause shrinkage or dispersion of tissue cell structures, damaging the normal tissue cell architecture. Therefore, slides should be air-dried or spin-dried before baking.
Improving Antigen Retrieval Methods
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Increase the volume of retrieval solution so that the liquid level is 3-5 cm above the slide rack, preventing repeated冲刷 of tissue by boiling liquid, which can cause detachment.
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Heat the retrieval solution to boiling, then add it to a covered staining jar. Place the slides into the staining jar, and then put both the jar and slides into a pressure cooker to continue the operation.
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Allowing slides to cool naturally after antigen retrieval can reduce detachment.
Anti-detachment Tips
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Special tissues require special treatment; for example, tissues rich in fat may need degreasing when necessary.
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During endogenous peroxidase inactivation, pay attention to the concentration of hydrogen peroxide; too high a concentration can easily cause bubbles and lead to detachment.
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Do not spin slides too vigorously; use tissue paper to absorb moisture from the edges.
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When washing slides with PBS buffer, try to use the immersion method.
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Perform bluing in a weakly alkaline environment using warm water or PBS buffer at pH 7.2-7.4.
References:
[1] , Chen Shunping, Zhu Yulian, Zheng Yun, et al. Discussion on the relationship between baking temperature and time for routine paraffin pathological tissue sections in HE staining [J]. Journal of Diagnostic Pathology, 2013(02):61.
[2] Zhang Rui, Ma Jianbo. Some experiences in preventing slide detachment in immunohistochemical staining [J]. Journal of Diagnostic Pathology, 23(5):2016