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Immunofluorescence (IF) microscopy is essential for studying protein expression and localization. Multiplex IF enables sequential staining of the same tissue section for comprehensive analysis but often involves harsh treatments or expensive reagents that can damage tissues and reduce signal quality. Here, we present a detailed protocol for multiplex IF using indirect antibody labeling, a mild stripping buffer, and cost-effective reagents. Importantly, mounting paraffin-embedded tissue sections on gelatin-coated coverslips enhances Z-axis optical resolution in widefield imaging.

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Multiplex Immunofluorescence Staining of Paraffin-Embedded Tissue: Coverslip Mounting of Tissue Sections Improves Z-axis Optical Resolution in Widefield Microscopy

  • 实验方案

  • 发表日期：

    2026年

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  • Gitte A. Pedersen,
  • Søren H. Elsborg,
  • Rikke Nørregaard,
  • Lene N. Nejsum

摘要

Immunofluorescence (IF) microscopy is essential for studying protein expression and localization. Multiplex IF enables sequential staining of the same tissue section for comprehensive analysis but often involves harsh treatments or expensive reagents that can damage tissues and reduce signal quality. Here, we present a detailed protocol for multiplex IF using indirect antibody labeling, a mild stripping buffer, and cost-effective reagents. Importantly, mounting paraffin-embedded tissue sections on gelatin-coated coverslips enhances Z-axis optical resolution in widefield imaging.