School activities
School, day 1 December 9th, 2019
UNIT 1: Fundamentals of electron microscopy. Brief historical review of electron microscopy. Concept of resolution. Components of an electron microscope. Description of the transmission electron microscope. Sample-beam interaction of electrons. New technologies in transmission microscopes.
(9-10:30 a.m.)
Coffee-Break
Practical activity 1
Each school participant will present the research is conduced and the main aims of his/her laboratory
(11-12 p.m.)
Practical activity 2
Concepts on Cell Biology: Mitochondrial Architecture. Parameters to analyze mitochondrial physiology and dynamics.
Example: Oxidative stress and mitochondrial adaptive shift during pituitary tumor growth.
Activity for the participants: Experimental design to be presented on the last day of the School.
(2-6 p.m.)
Instructors/Speakers
Ana Lucia De Paul, Universidad Nacional de Córdoba, Córdoba, Argentina
Liliana Sosa, Universidad Nacional de Córdoba, Córdoba, Argentina
Alexandra Latini, Universidade Federal de Santa Catarina, Florianópolis, Brasil
School, day 2 December 10th, 2019
UNIT 2: Preparation of biological samples for transmission electron microscopy (TEM). Fixation. Types of fixation and samples. Factors affecting fixation: temperature, duration, pH, osmolarity. Resin types: epoxy and methacrylate. Contrast principles. Introduction to ultramicrotomy. Block sculpture. Getting semifinished and thin cuts. Problems in cutting with the ultramicrotome. Immunocytochemistry. Principle of the antigen-antibody reaction. Criteria for conducting immunocytochemistry for TEM: antibody specificity and affinity, accessibility, antigenicity and preservation of the ultrastructure. Types and uses of controls. Problems in immunocytochemistry. Sensitivity. Types of immunostaining: Pre and post-incorporation. Double immunocytochemistry.
(9-10:30 a.m.)
Coffee-Break
Practical activity 3
Each school participant will present the research is conduced and the main aims of his/her laboratory
(11-12 p.m. and 2-5 p.m.)
Instructors/Speakers
Ana Lucia De Paul, Universidad Nacional de Córdoba, Córdoba, Argentina
Liliana Sosa, Universidad Nacional de Córdoba, Córdoba, Argentina
Alexandra Latini, Universidade Federal de Santa Catarina, Florianópolis, Brasil
Eliana Medeiros, Universidade Federal de Santa Catarina, Florianópolis, Brasil
Ramiro Zepeda, Universidad de Talca, Santiago, Chile
School, day 3 December 11th, 2019
Practical activity 4
Immunocytochemistry performed in sections of LR-White. Choice of secondary antibody. Handling of crates. Use of controls. Tokuyasu technique: Inclusion protocols. Manipulation of the cryo-ultra-microtome. Preparation of the samples and freezing. Sections picking-up, staining and observation under an optical microscope. Preparation of grids with Formvar film.Immunocytochemistry in cryosections. Contrasting with uranyl and methylcellulose film. Immunofluorescence practice. Observation of immunocytochemistry performed in fluorescence and electron microscopy. Comparison of different techniques. Interpretation of images.
(9-12 a.m.)
UNIT 3A: Immunocytochemistry. Principle of the antigen-antibody reaction. Criteria for conducting immunocytochemistry for TEM: antibody specificity and affinity, accessibility, antigenicity and preservation of the ultrastructure. Types and uses of controls. Problems in immunocytochemistry. Sensitivity. Types of immunostaining: Pre and post-incorporation. Double immunocytochemistry. Cold fixation and Tokuyasu technique. Fixation of the sample: high pressure and low temperature techniques (high pressure freezing), inclusion of samples at low temperature (Freeze Replacement).
(2-3:30 p.m.)
Coffee-Break
UNIT 3B: Tokuyasu technique and its applications. Cryoprotection and means of inclusion: sucrose and gelatin. Mounting and freezing. Cryo-ultra- microtomy: sculpture, obtaining of cuts and mounting in grids. Immunostaining and contrast.
(4-6 p.m.)
Instructors/Speakers
Ana Lucia De Paul, Universidad Nacional de Córdoba, Córdoba, Argentina
Liliana Sosa, Universidad Nacional de Córdoba, Córdoba, Argentina
Eliana Medeiros, Universidade Federal de Santa Catarina, Florianópolis, Brasil
Ramiro Zepeda, Universidad de Talca, Santiago, Chile
School, day 4 December 12th, 2019
UNIT 4: Immunofluorescence: basic concepts and their applications. Quantification. Correlation microscopy: CLEM. Introduction to CLEM. Tokuyasu cryosections for CLEM. Types of CLEM. Preparation of the sample and sectioning. Acquisition of images and overlapping. When to use CLEM. CLEM applications and examples.
(9-10:30 a.m.)
Coffee-Break
Practical activity 5
CLEM
(11-12 p.m.)
Instructors/Speakers
Ana Lucia De Paul, Universidad Nacional de Córdoba, Córdoba, Argentina
Liliana Sosa, Universidad Nacional de Córdoba, Córdoba, Argentina
Eliana Medeiros, Universidade Federal de Santa Catarina, Florianópolis, Brasil
Round table
Chair: Ramiro Zepeda, Universidad de Talca, Chile
Discussion with graduate students: Presentation of experimental designs
Participation of Louis-Éric Trudeau
Pharmacology and Physiology Department, University of Montreal, Montreal, Canada.
labotrudeau.org
(2-3 p.m.)