Nov 12, 2021
Collection of rat vagal tissue samples for TEM imaging
- 1Department of Psychological Sciences, Purdue University
- SPARCTech. support email: info@neuinfo.org
Protocol Citation: Terry Powley, Deborah Jaffey 2021. Collection of rat vagal tissue samples for TEM imaging. protocols.io https://dx.doi.org/10.17504/protocols.io.bzwcp7aw
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: November 08, 2021
Last Modified: November 12, 2021
Protocol Integer ID: 54948
Abstract
This protocol describes the methods used to generate samples of rat vagus tissue suitable for TEM imaging.
Protocol materials
Sprague-DawleyEnvigo
2018 Teklad global 18% protein rodent dietEnvigo
XylazineAkorn IncCatalog #NDC: 59399-110-20
Paraformaldehyde BAKER J.T. BakerFisher ScientificCatalog #02-003-576
Glutaraldehyde 50% EM Grade AqueousElectron Microscopy SciencesCatalog #16320
KetaminePatterson VeterinaryCatalog #07-803-6637
Animals
Animals
Sprague-DawleyEnvigo
rats (2–4 months old, male and female) were housed in shoe-box cages with bedding material in an Association for Assessment and Accreditation of Laboratory Animal Care-approved colony room, temperature (22–24°C) and humidity (40%–60%) controlled. The room was maintained on a 12:12 hour light–dark schedule. Pelleted chow
2018 Teklad global 18% protein rodent dietEnvigo
and filtered tap water were provided ad libitum. All husbandry practices conformed to the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals (8th edition) and were reviewed and approved by the Purdue University Animal Care and Use Committee. All efforts were made to minimize any suffering as well as the number of animals used.
Perfusion
Perfusion
Animals were perfused according to the following procedure. Specifically, they were overdosed with anesthetic (intraperitoneal injection of ketamine/xylazine [275 mg/kg of ketamine/27.5 mg/kg of xylazine]):
KetaminePatterson VeterinaryCatalog #07-803-6637
XylazineAkorn Animal HealthCatalog #NDC: 59399-110-20
Animals were then exsanguinated with fresh physiological saline, and then transcardially perfused with a fresh solution of EM-grade fixatives (2% paraformaldehyde/1.25% glutaraldehyde) at 4°C:
Paraformaldehyde BAKER J.T. BakerFisher ScientificCatalog #02-003-576
Glutaraldehyde 50% EM Grade AqueousElectron Microscopy SciencesCatalog #16320
in 0.1 M phosphate-buffered saline ([PBS], pH = 7.3) for 20 minutes.
Dissection and Fixation
Dissection and Fixation
Nerve bundles, trunks, and branches were immediately dissected out, manipulating the vagus as little as possible, removing only the minimum amount of connective tissue or fat require to access and remove the samples (4mm long sections at each sampled level) and moved into the same fixative combination as in step 2 overnight at 4°C on an oscillating platform.
The following morning, the tissue specimens were rinsed (3 × 30 min at 4°C) in PBS, transferred to individual shipping vials under PBS, and shipped overnight to the TEM laboratory for further processing and microscopy - see the protocol below:
Protocol
NAME
Nerve tissue processing for transmission electron microscopy (TEM)
CREATED BY
Natalia Biscola