Journal Day3 histopathology

Research article

A microwave method for plastic embedding of nervous tissue for light and

electron microscopy

Evan Calkins

, Edvinas Pocius

, Gail Marracci

, Priya Chaudhary

Department of Neurology, L226, Oregon Health &Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97239, United States

Research, Department of Veterans Affairs Medical Center, 3710 SW U.S. Veterans Hospital Road, Portland, OR 97239, United States

ARTICLE INFO

Keywords:

Cell biology

Neuroscience

Pathophysiology

Pathology

Neurology

Ophthalmology

Spinal cord

Optic nerve

Plastic embedding

Electron microscopy

Microwave

ABSTRACT

Background: Fast, effective, and rapid processing of central nervous system (CNS) tissue with good preservation of

myelin, especially in tissue from diseased mice, is important to many laboratories studying neurosciences.

New method: In this paper, we describe a new method to process and embed CNS tissue from mice. Spinal cords

and optic nerves from naive C57BL/6 mice were used to standardize the microwave protocol following perfusion

with ﬁxative. The CNS tissue was processed and embedded using the microwave embedding protocol.

Results: We observed that the tissue is well preserved and good quality light and electron microscope images were

obtained after using the microwave embedding protocol.

Comparison with existing methods: Traditional way of embedding CNS tissue in resin is challenging and time

consuming. The microwave technology offers an efﬁcient way to quickly embed CNS tissue while preserving

morphology and retaining the integrity of the myelin.

Conclusions: This new method is fast, reliable and an effective way to embed CNS tissue in resin.

1. Introduction

Many experimental murine models have been developed that use

central nervous system (CNS) tissue to study different neurodegenerative

diseases. Preservation of cells, organelles, axoplasm and myelin integrity

are important for understanding pathology. Conventional ﬁxation, pro-

cessing and resin embedding of tissue for light and transmission electron

microscopic examination is a slow, time consuming process that requires

days to complete (Spurr, 1969;Newman and Hobot, 1999). In recent

years, use of microwave technology for processing tissue has gained

popularity due to ease, superior visualization of morphology and short-

ening tissue processing time to a few hours (Giberson and Demaree,

1999;Giberson et al., 1997,2003). The microwave technology also al-

lows ﬂexibility where parts of the protocol can be done using the mi-

crowave and the ﬁnal resin inﬁltration can be in a conventional oven. In

this paper, we detail a protocol for processing, dehydrating, and

embedding of CNS tissue for light and electron microscopy (EM) using

the PELCO Biowave

Pro þmicrowave. This protocol preserves ultra-

structure, myelin, and tissue organelles, thereby saving many days of

processing compared to the conventional method.

2. Materials and methods

At least 29 small Petri dishes per tissue holder (Ted Pella, Redding,

CA; Cat. No. 36135-5)

3 large Petri dishes, polypropylene, 150mm (ProSciTech, Kirwan,

QLD, Australia; Cat. No. H636-150)

3–400ml beakers (Electron Microscopy Sciences (EMS), Hatﬁeld, PA;

Cat. No. 60974)

3–1000ml beakers (EMS, Hatﬁeld, PA; Cat. No. 60978)

3–20ml scintillation vials (EMS, Hatﬁeld, PA; Cat. No. 72632)

Ampoule cracker (EMS, Hatﬁeld, PA; Cat. No. 60600)

5ﬂ. oz. staccup beaker (EMS, Hatﬁeld, PA; Cat. No. 60950)

Acetone 500ml, Thermo Fisher Scientiﬁc, Pittsburgh, PA; Cat. No.

S25120B

PELCO Prep-Eze specimen holders (Ted Pella, Inc., Redding, CA; Cat.

No. 36158-1)

PELCO Prep-Eze numbered mat (Ted Pella Inc., Redding, CA; Cat. No.

36158-3)

Embedding Capsule Holder (EMS, Hatﬁeld, PA; Cat. No. 70022-04)

Capsule Press (EMS, Hatﬁeld, PA; Cat. No. 69920-00)

Acetone Wash Bottle, 250 ml (EMS, Hatﬁeld, PA; Cat. No. 64100)

* Corresponding author.

E-mail address: chaudhar@ohsu.edu (P. Chaudhary).

Contents lists available at ScienceDirect

Heliyon

journal homepage: www.cell.com/heliyon

https://doi.org/10.1016/j.heliyon.2019.e03036

Received 16 July 2019; Received in revised form 17 September 2019; Accepted 10 December 2019

nc-nd/4.0/).

Heliyon 6 (2020) e03036

Readings in the Philippine History (GEED 10033)

Polytechnic University of the Philippines

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