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P10. Penta-lung IHC staining

Jesper Lund Lauridsen, BLS, and Jess Pilegaard, MD

Department of Pathology, Regional Hospital Holstebro, Denmark

What is Pentalung?

  • A fast, tissue saving double staining technique with five antibodies applied on the same slide to be used for small lung biopsies

Double staining by IHC

  • MultiVision contains a cocktail of two mono-specific detection systems
  • One system is visualized with HRP the other with AP
  • MultiVision detects multiple antigens simultaneously in the same tissue sections (1)

Challenges of double staining

  • Two primary antibodies raised in same species e.g., mouse
  • Contrast between colours of the visualization systems and counterstaining
  • Solubility of the product in the mounting medium

MultiVision

  • The MultiVision contains 2 indirect polymer reinforcement systems
  • The polymer is made of small units of amino acids
  • Mono-specific: Goat anti-rabbit HRP+
  • Mono-specific: Goat anti-mouse AP+

Method

  • Incubation with primary ab
  • Incubation with MultiVision
  • Visualization of AP with LVBlue
  • Visualization of HRP with LVRed
  • Nuclear staining with Haematoxylin
  • Dehydration
  • Mounting with xylen based mounting medium

5 antibodies

To demonstrate squamous cell carcinoma:

  • p63, nuclear marker: Mouse monoclonal antibody
  • CK5 and CK14, cytoplasmic marker: Rabbit monoclonal antibody

To demonstrate adenocarcinoma:

  • TTF-1, nuclear marker: Rabbit monoclonal antibody
  • Napsin A, cytoplasmic marker: Mouse monoclonal antibody

Results with 5 antibodies

  • If squamous cell carcinoma: blue nuclei and red cytoplasm
  • If adenocarcinoma: red nuclei and blue cytoplasm

1) Emmy Yanagita, Naoko Imagawa, Chiho Ohbayashi, and Tomoo Itoh. Rapid Multiplex Immunohistochemistry Using the 4-antibody Cocktail YANA-4 in DifferentiatingPrimary Adenocarcinoma From Squamous Cell Carcinoma of the Lung. Appl Immunohistochem Mol Morphol 2011;19:509–513
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KONXION A/S