Quantifying immunogold label on EM thin sections
April 5th to 7th 2012
Host: House Research Institute
Location: House Research Institute
2100 W. 3rd St. Los Angeles, CA 90057
Organizer: Paul Webster, Ph.D.
Instructors: Terry Mayhew, Ph.D. & Paul Webster, Ph.D.
Registration Form
Course Flyer
An important tool in cell biology is the combination of immunogold labeling and transmission electron microscopy (TEM) by which target molecules of interest are bound specifically to affinity markers (primary antibodies) and then detected and localized with visualization probes (e.g. colloidal gold particles bound to protein A). Gold particles are electron dense and available in different sizes and so can be used also to quantify one or more defined targets in cell compartments. During the past decade, new ways of quantifying gold labeling within cells have been devised. Their efficiency and validity rely on sound principles of specimen sampling, event counting and inferential statistics. These include random selection of items at each sampling stage (e.g. specimen blocks, thin sections, microscopical fields), stereological analysis of cell ultrastructure, unbiased particle counting and statistical evaluation of a suitable null hypothesis (no difference in the intensity or pattern of labeling between compartments or groups of cells).
The following approaches have been developed:
[1] A target molecule can be tested for preferential labeling by mapping the localization of gold particles across a set of cell compartments.
[2] Data from wild-type and knockdown/knockout cells can be used to correct raw gold particle counts, estimate specific labeling densities and then test for preferential labeling.
[3] The same antigen can be mapped in two or more groups of cells to test whether or not there are experimental shifts in compartment labeling patterns.
[4] In multiple- labeling studies, different sizes of gold particle can be used to test whether or not different antigens co-localize in one or more compartments.
[5] Absolute numbers of gold particles can be mapped over compartments at specific positions within polarized, oriented or dividing cells.
The course will review the current state-of-the-art and approaches will be illustrated by means of practical exercises.
For more information expand a panel below for a provisional schedule, instructor biographies, and registration details:
Introduction to the course (Webster)
Introduction to quantification: general aspects of study design (Mayhew)
Preparing biological material for thin sectioning (Webster - Lecture)
Immunolabeling thin sections of biological material (Webster - Lecture)
Lunch
Microscopical sectioning and basic stereological ideas for event counting (Mayhew -Lecture)
The crucial importance of random sampling (Mayhew - Lecture)
Gold counting – overview of methods (Mayhew - Lecture)
Student discussion (question & answer session)
Method 1: Testing for preferential labellng of subcellular compartments (Mayhew - Lecture)
Method 1: Testing for preferential Labellng of subcellular compartments (Mayhew -Practical Exercise)
Student discussion (question & answer session)
Lunch
Method 2: Calculating specific labelling density (Mayhew - Lecture)
Method 2: Calculating specific labelling density (Mayhew - Practical Exercise)
Student discussion (question & answer session)
Methods 2 and 4: Testing for shifts in labeling and for co-localization (Mayhew - Lecture)
Methods 2 and 4: Testing for shifts in labeling and for co-localization (Mayhew - Practical Exercise)
Student Discussion (question & answer session)
Lunch
Method 5: Mapping gold distributions in 3D (Mayhew - Lecture)
Method 5: Mapping gold distributions in 3D (Mayhew - Practical Exercise)
Student Discussion (question & answer session)
PROFESSOR TERRY M MAYHEW
Terry Mayhew is an Emeritus Professor at the University of Nottingham (UK). He gained a BA Honors degree in Zoology (Oxford University, 1968) and PhD in Cell Biology (University of Sheffield, 1972). At Sheffield, he became interested in stereology, a set of sampling and estimation tools for deriving quantitative 3D structural information about organelles, cells, tissues and organs from simple counts made on 2D slice images. He moved to Aberdeen University in 1980 and became Head of the Department of Anatomy in 1989. In 1991, he moved to the University of Nottingham as Head of Human Anatomy & Cell Biology. He retired from full-time academic life in December 2009.
He has applied design-based stereology to various biological problems using slice images generated by light and electron microscopy, confocal microscopy, magnetic resonance imaging, etc. Biological research has focused on growth, transport, adaptation, turnover, angiogenesis and vascular remodelling in human and murine placenta. He has also developed new methods for quantitative analysis of the spatial distributions of nanoparticles (including immunogold particles) using transmission electron microscopy. He has published over 230 papers and invited reviews together with a co-edited book and a glossary of anatomical terms for medical undergraduates. He has served on the editorial boards of Analytical & Cellular Pathology, Image Analysis & Stereology, J Anatomy, J Neurocytology and Placenta and enjoyed collaborative links with colleagues in Austria, Australia, Brazil, Canada, China, Denmark, Germany, Norway, Switzerland, Turkey, USA and UK. He teaches regularly on sponsored research-based techniques courses in the UK and overseas (Americas, Asia and Europe). In 2001, the University of Nottingham presented him with a Lord Dearing award for outstanding contributions to the development of teaching and student learning.
Dr Paul Webster
Paul Webster is a scientist at the House Research Institute where he also directs the Ahmanson Advanced EM & Imaging Center, a shared imaging resource. He came to Los Angeles from the Yale School of Medicine in New Haven, where he established and ran an imaging facility for the School of Medicine. He was born and obtained his BSc Honors and Ph.D, in the United Kingdom (UK). He first left the UK to work at the European Molecular Biology Laboratory in Heidelberg, and then to work with Don W. Fawcett in Nairobi Kenya, where he studied the cell biology of African trypanosomes.
A specialist in electron microscopy and related specimen preparation methods, Paul Webster has used these skills to study the basic mechanisms of how pathogens interact with mammalian cells. Most recently he has been studying bacterial biofilms and their relevance to human middle ear infections. He has published 120 papers and 10 book chapters on host-pathogen interactions, and serves on the editorial board of Microscopy Today, Microscopy Research & Technique and Cell Biology International. For many years he was a co-organizer of annual EMBO Stereology and Electron Microscopy in Cell Biology Practical Courses, and remains a full-time instructor and guest speaker on these courses.
Course Fees:
Before March 1st 2012 $900.00
After March 1st 2012 $1,200.00
Checks or Purchase Orders should be made out to: House Research Institute
Applications should be sent to:
Paul Webster, Ph.D.
House Research Institute
2100 West Third Street
Los Angeles
CA 90057
USA
Email: pwebster@hei.org
The application should include:
- A check or purchase order with the full course fee amount
- Full name and contact details of participant
- Name of organization sponsoring the participant
This course is expected to be very popular so early registration is encouraged.
