Sep 20, 2021
Nerve tissue processing for transmission electron microscopy (TEM)
- 1Department of Neurology, Icahn School of Medicine at Mount Sinai, New York, NY, USA;
- 2Departments of Neurology and Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA and Neurology Service and RR&D National Center for the Medical Consequences of Spinal Cord Injury, James J. Peters Veterans Administration Medical Center, Bronx, NY, USA
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Natalia Biscola, Leif Havton 2021. Nerve tissue processing for transmission electron microscopy (TEM). protocols.io https://dx.doi.org/10.17504/protocols.io.xpxfmpn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 01, 2019
Last Modified: September 20, 2021
Protocol Integer ID: 19927
Keywords: TEM, embedding, plastic resin, morphometric analysis
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 μm) are obtained and stained with 1% toluidine blue solution. Light microscopy image capturing is performed at 100X magnification for detailed analysis.
Materials
MATERIALS
Propylene oxideElectron Microscopy SciencesCatalog #20401
Eponate 12 kit with DMP-30Ted Pella Inc.Catalog #18010
37% Formaldehyde Merck MilliporeSigma (Sigma-Aldrich)Catalog #252549
GlutaraldehydeTed Pella Inc.Catalog #18431
Ethyl Alcohol 200 ProofGold Shield Distributors
Osmium TetroxideElectron Microscopy SciencesCatalog #19120
Sodium phosphate monobasicVWR International (Avantor)Catalog #0823-2.5KG
Sodium phosphate dibasicCatalog #0348-2.5KG
Sodium chlorideVWR International (Avantor)Catalog #BDH9286-2.5KG
Toluidine blueElectron Microscopy SciencesCatalog #22050
DPX Mounting MediumElectron Microscopy SciencesCatalog #13510
Safety warnings
Osmium tetroxide (OsO4) is VERY TOXIC. Work in the fume hood and use appropriate PPE.
nerve tissue collection
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
00:20:00
After dissection, the tissues of interest are postfixed in the same fixative overnight at 4°C and then rinsed 3 times with PBS for 00:30:00 each
Wash tissues 3 times with ddH2O for 00:10:00 each
Fixation
Fixation
Fix tissues with 1% Osmium solution diluted in ddH2O for 01:00:00 (Place vials with tissue +Osmium solution in circular shaker)
Safety information
*Osmium is VERY TOXIC. Work in the fume hood and use appropriate PPE.
Note
Prepare Osmium solution at least one day in advance.
Wash tissues 3 times with ddH2O for 00:10:00 each
Dehydration
Dehydration
Dehydrate tissues with alcohol solutions in different concentrations, as follow: 30%, 50%, 70, 80, 95, and 100% (twice) solution diluted in ddH2O 00:10:00 each
Note
Use 200 Proof Alcohol to prepare the solutions.
Continuing the dehydration place 100% propylene oxide in the tissues for 00:20:00
Infiltration
Infiltration
Infiltration step starts with tissues in 50% propylene oxide+ 50% Epon solution for 2 hours. After, place 100% Epon resin in vials overnight.
Note
Epon Solution:
Use Eponate 12 Kit, with DMP-30 (Ted Pella #18010).
To prepare 100mL Epon: Mix 47.6mL of RESIN; 32.0mL of DDSA; 23.0mL of NMA; and 1.6mL of DMP-30
Embedding
Embedding
Block tissue of interest in plastic molds. Place the tissue in a direction to obtain cross sections later.
Semi-thin sectionning
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 00:00:30
Wash sections with ddH2O, dry and coverslip with DPX medium.
Image acquisition
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