Nerve tissue processing for transmission electron microscopy (TEM)
Natalia Biscola, Leif Havton
Abstract
Transmission electron microscopy (TEM) studies promote an improved understanding of functional studies and provide critical ultrastructural information. This document provide a step by step protocol to embed tissues for TEM analysis. First, the animals are transcardially perfused with 2% paraformaldehyde and 1.25% glutaraldehyde diluted in 0.12M Millonigs buffer (MB), pH 7.3, for 15-20 minutes. After dissection, the tissues are postfixed in the same fixative overnight at 4°C and then rinsed in MB 3 times for 30 minutes each. Embedding protocol includes osmication, dehydration, and embedding of tissues in plastic resin. The tissue blocks are trimmed and semithin sections (0.5 μ m) are obtained and stained with 1% toluidine blue solution. Light microscopy image capturing is performed at 100X magnification for detailed analysis.
Before start
Steps
nerve tissue collection
Animals are transcardially perfused with 0.12M Millonigs buffer (MB), ph=7.3, followed by 2% paraformaldehyde and 1.25% glutaraldehyde diluted in MB for
0h 20m 0s
After dissection, the tissues of interest are postfixed in the same fixative overnight at 4°C and then rinsed 3 times with PBS for 0h 30m 0s
Wash tissues 3 times with ddH2O for 0h 10m 0s
Fixation
Fix tissues with 1% Osmium solution diluted in ddH2O for 1h 0m 0s (Place vials with tissue +Osmium solution in circular shaker)
Wash tissues 3 times with ddH2O for 0h 10m 0s
Dehydration
Dehydrate tissues with alcohol solutions in different concentrations, as follow: 30%, 50%, 70, 80, 95, and 100% (twice) solution diluted in ddH2O 0h 10m 0s
Continuing the dehydration place 100% propylene oxide in the tissues for 0h 20m 0s
Infiltration
Infiltration step starts with tissues in 50% propylene oxide+ 50% Epon solution for 2 hours. After, place 100% Epon resin in vials overnight.
Embedding
Block tissue of interest in plastic molds. Place the tissue in a direction to obtain cross sections later.
Semi-thin sectionning
Cut semi-thin cross section (0.5μm) and place them on glass slides.
Label sections with 1% toluidine blue solution diluted in ddH2O for 0h 0m 30s
Wash sections with ddH2O, dry and coverslip with DPX medium.
Image acquisition
Acquire and analyze images (10X magnification for oveview image and 100X magnification for detailed image) using a Nikon Eclipse E600 microscope and Nikon camera DS-Fi3
