IHC Technique Guide: Removing “Water Droplets” from Tissue Sections
Preface:
Qualified sections are the prerequisite for pathological diagnosis. Often, the prepared sections are not perfect, such as gaps resembling“water droplets” or punctate crystals and dark nuclei due to insufficient dehydration or poor mounting with overly dry mounting medium, which greatly affects slide reading and mood. What to do?Here comes a little trick.
Reasons for water droplets or punctate crystals in sections
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Poor mutual solubility between clearing agent and dehydrating agent;
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Dehydrating agent failure or untimely replacement of dehydrating agent, resulting in insufficient dehydration;
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Weather conditions causing sections to become damp;
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Long time without mounting or overly dry mounting medium failing to completely cover the tissue, creating gaps for “water droplets”;
Repair of sections
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Degreasing problematic sections: Remove the coverslip (if the coverslip is already stuck, heat until the balsam melts and then remove), soak in xylene until the adhesive on the section is completely removed, then take out from xylene;
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Section hydration: Immerse the completely degreased sections sequentially into two jars of absolute ethanol, two jars of 95% ethanol, and one jar of 85% ethanol for gradient ethanol hydration. For immunohistochemically stained sections, 2 min per jar; for HE stained sections, 5 s per jar;
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Section dehydration and clearing again: Dehydrate the sections through gradient dehydration in one jar of 85% ethanol, two jars of 95% ethanol, and two jars of absolute ethanol; for immunohistochemically stained sections, 2 min per jar; for HE stained sections, 5 s per jar; clear in xylene for 2 min;
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Section remounting and observation: After clearing, directly mount the section with mounting medium and observe under the microscope.
If it is clearly known that the crystals and dark nuclei are caused by poor mounting, another quick treatment method is to heat the section and then directly soak it in xylene, remove the coverslip, and remount again.
References:
[1] Wu Zhenru, Chen Menglin, Li Li, et al. A method for removing water droplets from pathological sections[J]. Journal of Clinical and Experimental Pathology, 2021, 37(2): 236-237. DOI:10.13315/j.cnki.cjcep.2021.02.028.
[2] Gu Liping, Yu Jixia. Discussion on air crystals and treatment methods in routine section preparation[J]. Journal of Clinical and Experimental Pathology, 2020, 36(3): 366-367. DOI:10.13315/j.cnki.cjcep.2020.03.032.
(02):236-237.[doi:10.13315/j.cnki.cjcep.2021.02.028]