We describe here a simple technique for flat-osmicating and flat-embedding of immunolabelled vibratome sections. The technique is particularly useful for large specimens such as whole cross-sections of rat spinal cord. After the vibratome section has been flat-osmicated on a flat surface under a glass cover slip, it is dehydrated and then embedded by placing it in a small drop of epoxy resin on a flat base of polypropylene plastic and overlaying a small square piece of cellulose acetate cut from heat-resistant overhead projector transparency film for photocopying. The thin layer of resin containing the flat-embedded vibratome section is then separated from the base and glued onto the flat end of a pre-polymerised blank block of resin. The method produces flat-embedded vibratome sections and thus allows serial large uniformly labelled semithin and ultrathin sections to be obtained of the whole cross-section of the rat spinal cord. This facilitates the observation and quantification of labelled cells in the specimen. Because of its simplicity the technique also allows one worker to process more than 100 vibratome sections at the one time

Dalton

DeFelipe

Friedman

Glauert

Grimley

Holländer

Kim B. Nguyen

Michael P. Pender

Pender

Priestley

Schwartz

Valentijn

Nguyen, Kim B.

Pender, Michael P.

University of Queensland eSpace

Journal of Neuroscience Methods, 65 (1), 1996, pp. 51-54. 
DOI http://dx.doi.org/10.1016/0165-0270%2895%2900145-X 
A Simple Technique For Flat Osmicating And 
Flat Embedding Of Immunolabelled Vibratome 
Sections Of The Rat Spinal Cord For Light And 
Electron Microscopy  
Kim B. Nguyen and Michael P. Pender  
 
Neuroimmunology Research Unit, Department of Medicine, University of Queensland, Royal Brisbane 
Hospital, Brisbane, Australia. 
 
Abstract 
We describe here a simple technique for flat-osmicating and flat-embedding of immunolabelled vibratome 
sections. The technique is particularly useful for large specimens such as whole cross-sections of rat spinal 
cord. After the vibratome section has been flat-osmicated on a flat surface under a glass cover slip, it is 
dehydrated and then embedded by placing it in a small drop of epoxy resin on a flat base of polypropylene 
plastic and overlaying a small square piece of cellulose acetate cut from heat-resistant overhead projector 
transparency film for photocopying. The thin layer of resin containing the flat-embedded vibratome section 
is then separated from the base and glued onto the flat end of a pre-polymerised blank block of resin. The 
method produces flat-embedded vibratome sections and thus allows serial large uniformly labelled semithin 
and ultrathin sections to be obtained of the whole cross-section of the rat spinal cord. This facilitates the 
observation and quantification of labelled cells in the specimen. Because of its simplicity the technique also 
allows one worker to process more than 100 vibratome sections at the one time. 
 
Keywords: flat-osmicating; flat-embedding; vibratome section; immunolabeling 
 
 
1. Introduction  
 
The pre-embedding immunolabelling technique has provided a useful method to study various 
tissues and, in particular, the diverse cellular populations of the nervous system. It enables the 
labelling of glial and neuronal cells as well as infiltrating inflammatory cells in the central 
nervous system in pathological conditions such as experimental autoimmune encephalomyelitis 
(Pender et al., 1992). In contrast to immunolabelling techniques using frozen or paraffin sections, 
the pre-embedding immunolabelling technique gives good preservation of tissue structure and 
allows high-resolution light microscopy. It also allows further detailed examination at electron 
microscopy. However, problems have been encountered with this technique. Because poor 
antibody penetration limits the reaction product to a thin layer at the surface of the vibratome 
maximal number of uniformly labelled semithin sections can be obtained from the shallow depth 
of useful tissue. Vibratome sections, which are usually 80-150 µm thick, tend to become 
undulated as a result of the constant stirring that is required during the immunolabelling 
procedure. Unless the section is kept flat during osmication, any undulation will become 
permanent as the tissue hardens. Even if the section is successfully flat-osmicated, it may become 
curved during resin polymerisation unless a flat-embedding technique is used. Quantitative 
studies of semithin sections cut from curved embedded vibratome sections are limited by the 
small area that is labelled in the plane of section.  
 
In our studies involving the identification and quantification of apoptotic cells in the spinal cord 
of rats with experimental autoimmune encephalomyelitis (Pender et al., 1992), we require 
completely flat vibratome sections with a diameter sometimes greater than 3 mm. A number of 
techniques employing different means to obtain flat-embedded sections have been described 
(Grimley, 1965; Holländer, 1970; Schwartz, 1982; Priestley, 1984; Yu and Schwartz, 1989: 
Valentijn et al., 1989; DeFelipe and Fairén, 1993).  
 
However, generally these techniques require special devices and/or involve many procedural 
steps and still do not produce completely satisfactory results. Most have either not mentioned or 
not described in detail how the fixation with OsO4, is performed. Post-fixing with this fixative is 
an essential step in the processing of nervous tissue and is also a crucial step that determines the 
flatness of the embedded vibratome section. Schwartz (1982) has described a technique for flat-
osmicating and flat-embedding of vibratome sections but this technique is difficult to perform. 
We have developed a simple technique that produces large flat-embedded vibratome sections and 
that allows one worker to process many sections at the same time.   
 
2. Materials and methods 
 
Vibratome sections (80-100 µm thick) were obtained from spinal cord tissue of rats that had been 
perfused through the ascending aorta with 4% paraformaldehyde, 0.05% glutaraldehyde in 
phosphate-buffered saline, pH 7.4. The vibratome sections were immunolabelled using various 
procedures and the efficacy of the labelling procedure was assessed by examining buffer-soaked 
sections under the light microscope. The sections are then washed with 0.9% saline in 5 ml vials, 
post-fixed with 2% paraformaldehyde, 2.5% glutaraldehyde/0.1 M sodium cacodylate buffer for 1 
h and washed again with 4 changes of saline, each for 10 min. The sections are still flexible at 
this stage. Groups of 5-10 floating sections are transferred with about 0.5 ml saline into the 
compartments of a white plastic slide tray (Bel-Art, USA) or into small plastic Petri dishes. Saline 
is carefully removed through a fine tip of a plastic transfer pipette or using the capillary action of 
filter paper. Still under the protection of a fumehood, a few drops of 1% OsO4, fixative (Dalton’s 
solution) (Dalton, 1955) are placed on the sections that are lying flat on the bottom of the slide 
tray. Glass coverslips are gently placed over each group of sections to keep the sections flat 
during osmication.  
 
After 30 min, a few drops of OsO4, fixative are added to the edges of the coverslips so that the 
coverslips float above the sections and can be removed with fine forceps. After being soaked for a 
further 5 min to enable the fixative to penetrate completely into the centres of the sections, the 
sections are gently collected with a fine brush into large vials with a flat bottom (e.g., 20 ml 
scintillation glass vials). The flat-osmicated sections are then washed with 5 - 10 changes of 
saline over at least 2 h, with gentle and only occasional stirring. They are stained en bloc with 1% 
uranyl acetate in saline for 1 h, washed with saline and dehydrated in 70% ethanol, 90% ethanol 
and 90% acetone followed by two changes of absolute acetone before being infiltrated 
sequentially with mixtures of acetone/epoxy resin in the proportions 3 : 1, 1: 1, and 1: 3 and 
finally with full resin in which they remain overnight at 4°C.    
 
 
Fig. 1.  
A: A square piece of cellulose acetate (cut from overhead projector transparency film for photocopying) is 
placed over the resin-covered flat-osmicated vibratome section. The resin spreads out to form a thin layer.  
B: After polymerisation in an oven at 60°C overnight the thin layer of resin that contains the vibratome 
section and that is still attached to the cellulose acetate square is lifted off the polypropylene base with fine 
forceps.  
C: The flat-embedded vibratome section is placed on a glass slide with the exposed face directed upwards. 
The exposed face is then glued to the flat end of a blank BEEM block. Finally the cellulose acetate square 
is peeled off. 
 
Epoxy resin is prepared by mixing 20 ml of ‘Epon’, 16 ml of DDSA (dodecenylsuccinic 
anhydride), 8 ml of MNA (methyl Nadic anhydride) and 1.3 ml of BDMA 
(bencyldimethylamine) (Glauert, 1991). The still flat infiltrated sections are individually 
transferred using the thinly flattened tip of a wooden stick, with minimum resin attached, to a 
small drop of fresh resin placed on a flat base of polypropylene, such as the bottom of a freezer 
storage box. A square (1 x 1 cm) piece of cellulose acetate (cut from heat-resistant overhead 
projector transparency film for photocopying) is placed over each resin-covered section, as 
illustrated in Fig. 1. To prevent the cellulose acetate square from curling, the temperature for 
polymerisation is not allowed to go above 60°C. The square is held down by the surface tension 
of the resin between the square and the polypropylene base. The vibratome section is thereby kept 
flat during polymerisation. As the resin binds only loosely to the polypropylene base, the thin 
layer of polymerised resin containing the section can be readily separated from the base while 
remaining attached to the cellulose acetate square.  
 
At this stage the embedded section can be examined light microscopically on a glass slide with 
the exposed face directed upwards, or the section can be trimmed to a small area of interest, if 
required. The exposed face is then glued with cyanoacrylate or epoxy adhesive to a blank block 
that has been prepared by polymerisation in a BEEM capsule in an inverted position with the cap 
removed and with the open end lying on a flat polypropylene surface. The cellulose acetate 
square is then simply peeled off and the block is ready for semithin and ultrathin sectioning.  3. 
Results and discussion Our new technique consistently produces large flat-embedded vibratome 
sections from which we can cut serial uniformly labelled semithin whole cross-sections of the rat 
spinal cord (Fig. 2A-C). We have successfully used this technique on vibratome sections with 
diameters of up to 3.5 mm. The production of large uniformly labelled sections facilitates the 
observation and quantification of labelled structures within the specimens. The technique is 
simple and allows one worker to process more than 100 sections at the same time. For electron 
microscopy, we generally cut ultrathin sections from the whole cross-section of spinal cord.   
 
 
 
Fig. 2. Light micrographs of 1 µm semithin sections from a flat-embedded vibratome section of the sacral 
spinal cord of a Lewis rat with experimental autoimmune encephalomyelitis. The vibratome section was 
immunolabelled with Rip monoclonal antibody (Friedman et al., 1989) and the avidin-biotin-peroxidase 
complex technique to identify oligodendrocytes. A: Low power in black and white showing that the 
immunolabelling of oligodendrocytes (arrows) is even throughout the whole cross-section of the spinal 
cord (not counterstained). B,C: Higher powers of a section counterstained with toluidine blue and showing 
specific labelling of oligodendrocyte cytoplasm (large arrows). Other cells such as a neurone (arrowhead) 
and inflammatory cells (small arrows) am not labelled. Bars: (A) = 100µm; (B) = 50 µm; (0 = 25 µm. 
 
In our technique the thickness of the layer of resin containing the embedded vibratome section 
varies automatically with the thickness of the vibratome section. The section is kept flat during 
polymerisation by the cellulose acetate square which is held down by the surface tension of the 
resin between the square and the polypropylene base. In contrast, some previously published 
techniques rely upon a preset thickness for the layer of resin (e.g., the thickness of a cellulose 
acetate sheet) (Holländer, 1970; Yu and Schwartz, 1989). If the vibratome section is thinner than 
the preset thickness, it is still able to curve during polymerisation; if it is thicker, it is crushed by 
the externally applied force. A key step in our new technique is the washing step after flat 
osmication. The sections need to be kept lying on the flat bottom of the washing vial for most of 
the time during washing. They should be stirred gently and only occasionally, as the hardening 
process continues for some time after osmication, and as vigorous or continuous stirring at this 
stage can cause distortion of the partly hardened sections. Even a slightly undulated osmicated 
section can not be made completely flat during embedding, as any flattening force may crack the 
section. Another important requirement for the production of good results is the use of a minimal 
amount of resin to embed the section before overlaying the cellulose acetate square. This ensures 
close apposition of the square and the vibratome section. In conclusion, we have described a 
simple new technique that consistently produces large flat-embedded vibratome sections for 
observation and quantification of immunolabelled structures at light and electron microscopy.   
 
Acknowledgements   
 
We are grateful to Dr. B. Friedman for giving us the Rip antibody. This work was supported by the 
National Health and Medical Research Council of Australia.   
 
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Friedman, B., Hockfield, S., Black, J.A., Woodruff, K.A. and Waxman, S.G. (1989) In situ demonstration of mature 
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A simple technique for flat osmicating and flat embedding of immunolabelled vibratome sections of the rat spinal cord for light and electron microscopy

Nguyen, KB

Pender, MP

A simple technique for flat osmicating and flat embedding of immunolabelled vibratome sections of the rat spinal cord for light and electron microscopy (vol 65, pg 51, 1996)

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A simple technique for flat osmicating and flat embedding of immunolabelled vibratome sections of the rat spinal cord for light and electron microscopy

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