In Situ Hybridization (ISH) Combined with Immunocytochemistry (ICC) Co-detection of Phosphorylated EGFR in A431 Cultured Cells

Methods Mol Biol. 2023:2593:213-220. doi: 10.1007/978-1-0716-2811-9_13.


Antibodies have been commonly used to study protein phosphorylation since the first phospho-specific antibody was described in 1981. Antibodies can be developed so that they specifically recognize phosphorylated areas of particular proteins. In situ hybridization (ISH) is the technique where specific RNA or DNA molecules can be detected in a single cell without the need for antibodies. Using ACD's integrated Co-Detection Workflow (ICW), we have developed a protocol to use phospho-specific antibodies in combination with ISH to show co-localization of EGFR mRNA and EGFR proteins phosphorylated at different sites in tumor cells. Our protocol has been used for multiplexing Y1086 phosphorylated EGFR, Y1068 phosphorylated EGFR, and EGFR RNA in A431 human epidermoid carcinoma cells.

Keywords: Fluorescence detection; ISH-ICC Integrated Co-Detection Workflow (ICW); Immunocytochemistry (ICC); In situ hybridization (ISH); Multiplexing; Phospho-EGFR antibodies; Phospho-antibodies.

MeSH terms

  • Antibodies*
  • Cells, Cultured
  • ErbB Receptors* / genetics
  • ErbB Receptors* / metabolism
  • Humans
  • Immunohistochemistry
  • In Situ Hybridization
  • RNA, Messenger / genetics


  • ErbB Receptors
  • Antibodies
  • RNA, Messenger
  • EGFR protein, human