Currently viewing: 20 - 30 June | 2007 |National University of Singapore | Singapore

Programme (pre)

The theory and practice of the following techniques will be covered:

1. Epoxy resin embedding and sectioning
   
   
2. Tokuyasu cryosection method for immunogold labelling and immunofluorescence at the LM level
   
   
3. Freeze substitution and low temperature embedding in methacrylate resins for structural analysis and/or immunogold labelling at the EM and for LM. It should be noted that although the main focus of the course is on EM, the techniques of state of the art LM on sections represent an intermediate step in resolution between the confocal (and living cell) and the EM level.
   
   
4. Antibody labelling and gold labelling technologies
   
   
5. Negative staining approaches (including immunolabelling)
   
   
6. Cryo EM of isolated particles  (optional)
   
   
7. EM evaluation and interpretation of data
   
   
8. Stereology

Tentative Lecture Programme

 

Day 1
15.30 - 1545	Introductory Remarks: Mary Ng
15.45 - 19.30	Student Powerpoint presentations (7 mins each)
19.30 - 20:00	Lab tour
20:00	Get-together / Welcome party
Day 2
09.00 – 09.15	Introduction (Gareth Griffiths)
09.15 – 09.45	Overview of course techniques (Paul Webster)
09.45 – 10.30	Principles of transmission EM (Norbert Roos)
10.30 – 10.50	Coffee break
10.50 – 11.35	Introduction to plastic embedding and sectioning (Herb Hagler)
11.35 – 12.30	Introduction to Cryosectioning (Paul Webster)
12.30 – 13.30	Lunch
13.30	Practicals
Day 3
09.00 – 09.45	Introduction and Practical details of Immunolabelling (Paul Webster)
09.45 – 10.45	Vitrification of water and cryo EM (Norbert Roos)
10.45 – 11.00	Coffee break
11.00 – 11.50	Freeze substitution for EM (Heinz Schwarz)
11.50 – 12.15	New Instruments for High Pressure Freezing and Freeze Substitution (Andreas Nowak, Leica Mikrosysteme)
12.15 – 13.00	Lunch
13.00 - 13.45	New Developments at the knife edge (Helmut Gnägi, Diatome Ltd)
13.45	Practicals
Day 4
09.00 – 09.45	Colloidal gold (John Lucocq)
09.45 – 10.45	Correlating EM and light microscopy (Heinz Schwarz)
10.45 – 11.00	Coffee break
11.00 – 11.45	Chemical fixation for EM (Gareth Griffiths)
11.45 – 12.30	Invited local Speaker
12.30 – 13.30	Lunch
13.3	Practicals
Day 5
09.00 – 09.45	Antibody production and usage (Heinz Schwarz)
09.45 – 10.30	Theory and pitfalls of immunolabelling (Gareth Griffiths)
10.30 – 11.00	Coffee break
11.00 – 11.45	Sampling for EM (John Lucocq)
11.45 – 12.45	Counting gold particles and relating them to cell compartments I (John Lucocq)
12.45 – 13.30	Lunch
13.3	Practicals
Day 6
10.00 – 11.00	Details of cryo-sectioning (incl. videos) Peter Peters
11.00 – 11.15	Coffee break
11.15 – 12.15	Counting gold particles and relating them to cell compartments II (Terry Mayhew)
12.15 – 13.00	Practical on gold labelling densities (Terry Mayhew/John Lucocq)
13.00 – 14.00	Lunch
Day 7
09.00 – 09.45	Cryo EM  tomography (Marek Cyrklaff)
09.45 – 10.30	Interpretation in EM (Gareth Griffiths)
10.30 – 11.00	Coffee break
11.00 – 12.00	Negative staining and replica techniques (Heinz Schwarz)
12.00 – 13.00	Lunch
13:30	Practicals
Day 8
09.00 – 10.00	The new gold counting methods (Terry Mayhew)
10.00 – 11.00	Practical on the new gold counting methods
11:00	Practicals
Day 9
9:00	Practicals all day
Day 10
9:00	Practicals and Final Discussion
Practical details We foresee the course being divided into two groups, 1) being complete beginners, 2) having people with at least some EM experience (e.g. Epon embedding and sectioning). Group 1 will learn the basics of epoxy resin embedding and sectioning as well as the Tokuyasu cryo section method for immunogold labelling. Group 2 will focus on the Tokuyasu method as well as cryo EM and tomography.

                            

 

 

 

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