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Multiplex Immunofluorescence (mIF) Made Easy

Recorded On: 10/15/2020


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IHC as a procedure to detect individual antibodies has been well established for many years. Even the detection of 2-3 targets has been possible using standard chromogen detection systems. The application of IF has expanded the capabilities of detecting a greater number of targets on a regular basis. Several procedures have been reported using indirectly labeled primary antibodies with fluors, or labeled with indirect reporter molecules, such as individual short DNA oligos, other molecules including biotin, digoxigenin, DNP; or other custom modifications. One of the IF methods widely used is the detection of antibody targets using the HRP / TSA-fluor system. It has advantages such as intensity and stability of the fluorescent product.  However, a big disadvantage of this system is the requirement of complete removal of the primary and secondary-HRP detection antibodies used in previous rounds of detection. This “stripping” step is typically performed with heat in a retrieval solution. Thus, successive reheating of the stained tissue can compromise its integrity and morphology. This type of detection has been successfully established for manual applications and on automated staining instruments. In this presentation, we will describe a multiplexing approach developed in our laboratory using an innovative stripping method. Our approach has enabled the multiplexing of up to 5-7 markers that can be used to assess the tumor microenvironment (TME). Antibody panels used as models included a melanoma panel: CD3, CD8, GZMB, SOX10, Melan A; colon panel: GZMB, CDX2, CD3, PanCK+ and a lung SqCC panel comprised of: p40, CK 5-6, CK7, TTF-1, Cytokeratin HMW will be discussed.  This workshop is intended for those that have experience with multiplexing techniques in their laboratories and would like to learn more about multiplex IF made easy.

CEUs: This histology course is worth 1 continuing education credit. Course is available for 365 days from date of purchase. 

Julio Masabanda, PhD

Sr. Molecular Scientist

Biocare Medical

Julio Masabanda has over 25 years experience in Molecular Cytogenetics and Histology in Biotech and Academic laboratories both in Europe and North America. Julio's expertise includes genome analysis of farm, lab animals and humans.For the past 4 years at Biocare Medical, actively involved in R&D in Molecular Histology and Cytogenetics, developing reagents and technologies for detection of protein and nucleic acid targets on automatic staining platforms, including Fast-FISH, tissue conditioning and for multiplex immnunofuorescence.Julio has a Ph.D.in Comparative Genomics from Brunel University London, UK and a M.Sc., Biochemistry from Kharkiv National University, Ukraine.

Joseph Vargas, HTL(ASCP)

Director

Biocare Medical, LLC

Joe Vargas has twenty five years of experience working in academic, biotech and government laboratories. His areas of expertise are in immunohistochemistry, immunofluorescence, in situ hybridization, digital imaging and electron microscopy. He has worked for Biocare Medical in Concord, California for the past nine years. He's been focused on fluorescence imaging, managing projects involving animal polymer detections, in situ hybridization and providing technical support. Joe has a B.S. in Clinical Science and a M.S. in Biomedical Laboratory Science from San Francisco State University. He also has a certificate in Biological Electron Microscopy from San Joaquin Delta College and a certification as an HTL by the ASCP.

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Multiplex Immunofluorescence (mIF) Made Easy
Open to view video.
Open to view video. Objectives: Antibody panels and what they specifically target within the tumor microenvironment (TME); Methods of multiplexing immunofluorescence (mIF) on the bench and on automated platforms; A new approach to mIF that will allow attendees to expand on new antibody panels of their choice
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2 Questions Please complete the survey to receive your CEU certificate.
Certificate
1.00 CEUs credit  |  Certificate available
1.00 CEUs credit  |  Certificate available