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Simple method for staining and preserving epoxy resin-embedded animal tissue sections for light microscopy
LiYe Sciences Pol . 4, pp . 1839-1841, 1965 . Printed in Great Britain.
Pergamoa Press Ltd.
SIMPLE METHOD FOR STAINING ADTD PRESERVING EPOXY RESINEMBEDDED ANIMAL TISSUE SECTIONS FOR LIGHT MICROSCOPY K. W. Jeon Biology Department, Middlesex Hospital Medical School, London W. 1, England . (Eeceived 13 July 1965) While studying the fine structure of isolated specialised cells contained in relatively large blocks of embryonic tissues, it was found
necessary to examine many thick sections under a light microscope before
selecting suitable areas for thin-sectioning. them stained rapidly .
It was further desirable to have
Various methods have been used for staining thick
sections of tissues embedded for electron microscopy (see Pease (1) for references), either ßor mere identification of tissues or for refined microscopy .
However, all of them require a minimum of 5-10 minutes
before sections can be examined, and a simpler procedu re for rapid staining had to be devised.
By the method described below, it is possible to stain
resin-embedded sections for light microscopy in less than a minute .
Further, if desired, the sections can be mounted quickly for permanent preservation .
In addition, the staining quality is excellent and the sections
are particularly suitable for general histological purposes .
The staining procedure is to float thick sections (0 . 3-2F~), cut with glass or diamond knives, on a small drop of stain solution applied thinly on a clean slide, and to heat the slide on a hot plate (70-80 °) until the solution has evaporated .
As the solution gets hot, the sections become flattened and
adhere to the slide as they dry .
Under these conditions, small drops of
stain solutions usually take less than half a minute to evaporate, and the
sections can be examined immediately under a light microscope, including examination under oil immersion.
The stain found moat satisfactory for epoxy resin-embedded sections
was 0 . 1b0. 5% Azure II (G . T. Gurr Ltd. , London) dissolved in 1% borax solution . Azure II is not a specific stain for any structural components of
animal cells, but the staining density generally corresponds to their electron 1839
1840
STlI2âG TSICE 6EQTIOAB
Yol . 4, Ho . 19
FIfi . 1 Electron (a) and photo (b) micrographs of adjacent sections of 15 day-old mouse embryonic seminiferous tubule embedded in Araldite, to show the correlation between the electron density and Azure II-staining density of cell components . The tissue was fixed for 2 hr in cold 3% glutaldehyde buffered with veronal at pH 7 . 4 (2), and later osmicated for 1 hr . The section in (a) was double stained with uranyl acetate and lead citrate, and the 0 . 5 ~ section in (b) was stained with Azure II as described in the text . The large densely stained cells are the primordial germ cells (3) .
Yol . 4, Ro . 19
BTAIIIIFG THICE BECTIORB
1841
density (Fig. 1) .
The density of staining can be controlled both by the
concentration of the stain solution and by the amôunt placed on the elide.
It
has been found convenient to keep filtered Azure II solutions in small polyethylene bottles with nozzles, from which the solutions can be directly applied to and spread on a slide.
By using these bottles, xhe stain solutions can be
kept dust-free . The preparations can be kept without additional treatment for later
examination .
However, when preferred, the sections may be mounted with
a small amount of colophonium turpentine immediately, or after rinsing in cool distilled water to remove excess dye on the slides .
After rinsing,
the slides are dried with soft tissue paper and placed on a hot plate for another short period and cooled before mounting.
The mounted sections
should be dried at room temperature, as wrinkling occurs when dried at higher temperatures .
Dilute colophonium turpentine is available
commercially, but it was found better to prepare the mountant by dissolving colophony resin in turpentine oil to saturation at room temperature, for the
latter dries more quickly.
The mounting medium used here fulfils two requirements :
it
preserves the water-soluble dyes in the stained tissues, and keeps the resin sections unwrinkled .
Glycerine jelly was found to de-stain the sections in
a day or two, and all mounting media containing xylene or alcohols caused wrinkling of the resin sections . Although Araldite mixtures may also be
used Por mounting, they are far less practicable than colophonium turpentine for daily use .
The present method is moat useful for quick identification of tissues
before thin-sectioning, but it can also be used effectively for preparing histological sections from resin-embedded tissues .
The author wishes to thank Professor D, R. Newth very much for his "kind intérest and help during the course of this work . References 1. 2. 3.
D, C, PEASE, Histological techniques for electron microscopy . (2nd Edn. ), Chapter 7 . Academic Press, New York (1964) .
G. E, PALADE, J. Exptl. Med . 95, 285 (1952) .
K, W, JEON and D, R, NEWTH, in preparation (1965) .
Pergamoa Press Ltd.
SIMPLE METHOD FOR STAINING ADTD PRESERVING EPOXY RESINEMBEDDED ANIMAL TISSUE SECTIONS FOR LIGHT MICROSCOPY K. W. Jeon Biology Department, Middlesex Hospital Medical School, London W. 1, England . (Eeceived 13 July 1965) While studying the fine structure of isolated specialised cells contained in relatively large blocks of embryonic tissues, it was found
necessary to examine many thick sections under a light microscope before
selecting suitable areas for thin-sectioning. them stained rapidly .
It was further desirable to have
Various methods have been used for staining thick
sections of tissues embedded for electron microscopy (see Pease (1) for references), either ßor mere identification of tissues or for refined microscopy .
However, all of them require a minimum of 5-10 minutes
before sections can be examined, and a simpler procedu re for rapid staining had to be devised.
By the method described below, it is possible to stain
resin-embedded sections for light microscopy in less than a minute .
Further, if desired, the sections can be mounted quickly for permanent preservation .
In addition, the staining quality is excellent and the sections
are particularly suitable for general histological purposes .
The staining procedure is to float thick sections (0 . 3-2F~), cut with glass or diamond knives, on a small drop of stain solution applied thinly on a clean slide, and to heat the slide on a hot plate (70-80 °) until the solution has evaporated .
As the solution gets hot, the sections become flattened and
adhere to the slide as they dry .
Under these conditions, small drops of
stain solutions usually take less than half a minute to evaporate, and the
sections can be examined immediately under a light microscope, including examination under oil immersion.
The stain found moat satisfactory for epoxy resin-embedded sections
was 0 . 1b0. 5% Azure II (G . T. Gurr Ltd. , London) dissolved in 1% borax solution . Azure II is not a specific stain for any structural components of
animal cells, but the staining density generally corresponds to their electron 1839
1840
STlI2âG TSICE 6EQTIOAB
Yol . 4, Ho . 19
FIfi . 1 Electron (a) and photo (b) micrographs of adjacent sections of 15 day-old mouse embryonic seminiferous tubule embedded in Araldite, to show the correlation between the electron density and Azure II-staining density of cell components . The tissue was fixed for 2 hr in cold 3% glutaldehyde buffered with veronal at pH 7 . 4 (2), and later osmicated for 1 hr . The section in (a) was double stained with uranyl acetate and lead citrate, and the 0 . 5 ~ section in (b) was stained with Azure II as described in the text . The large densely stained cells are the primordial germ cells (3) .
Yol . 4, Ro . 19
BTAIIIIFG THICE BECTIORB
1841
density (Fig. 1) .
The density of staining can be controlled both by the
concentration of the stain solution and by the amôunt placed on the elide.
It
has been found convenient to keep filtered Azure II solutions in small polyethylene bottles with nozzles, from which the solutions can be directly applied to and spread on a slide.
By using these bottles, xhe stain solutions can be
kept dust-free . The preparations can be kept without additional treatment for later
examination .
However, when preferred, the sections may be mounted with
a small amount of colophonium turpentine immediately, or after rinsing in cool distilled water to remove excess dye on the slides .
After rinsing,
the slides are dried with soft tissue paper and placed on a hot plate for another short period and cooled before mounting.
The mounted sections
should be dried at room temperature, as wrinkling occurs when dried at higher temperatures .
Dilute colophonium turpentine is available
commercially, but it was found better to prepare the mountant by dissolving colophony resin in turpentine oil to saturation at room temperature, for the
latter dries more quickly.
The mounting medium used here fulfils two requirements :
it
preserves the water-soluble dyes in the stained tissues, and keeps the resin sections unwrinkled .
Glycerine jelly was found to de-stain the sections in
a day or two, and all mounting media containing xylene or alcohols caused wrinkling of the resin sections . Although Araldite mixtures may also be
used Por mounting, they are far less practicable than colophonium turpentine for daily use .
The present method is moat useful for quick identification of tissues
before thin-sectioning, but it can also be used effectively for preparing histological sections from resin-embedded tissues .
The author wishes to thank Professor D, R. Newth very much for his "kind intérest and help during the course of this work . References 1. 2. 3.
D, C, PEASE, Histological techniques for electron microscopy . (2nd Edn. ), Chapter 7 . Academic Press, New York (1964) .
G. E, PALADE, J. Exptl. Med . 95, 285 (1952) .
K, W, JEON and D, R, NEWTH, in preparation (1965) .