Transmission Electron Microscopy and Serial Sections
for Light Microscopy from the Same Block
in Routine Renal Pathology
ERNESTO O. HOFFMANN, M.D., HERMAN B. GARRETT II, M.S.,
JOAN R. COOVER, MT, B.S.(ASCP), EMSA, AND TERESA R. FLORES, HT(ASCP)
Routine serial sections for light microscopy (LM) from one
sample and transmission electron microscopy (TEM) from selected areas of another sample are current procedures in renal
pathology. At the present time, there are methods available
to perform high-resolution light microscopy (HRLM) and
TEM from the same block. However, this technology is not
directly applicable or convenient for routine renal biopsies.
Most methods presently available to obtain TEM studies from
different areas of the same large HRLM block use reembedding methods, which considerably delay TEM studies, or mesa
technics that fragment and waste the original block. Present
methods to obtain serial sections from plastic blocks have been
used in plant and animal morphology and need to be modified
for renal pathology.
In the present article, the authors present a technic that
produces immediate TEM sections from different areas of the
same HRLM block. This method does not produce fragmentation or waste of the original block, and TEM sections are
obtained within two hours after large HRLM sections are
screened. The latter sections are mounted serially on a regular
microscopic slide on 8-15 separate wells painted with waterrepellent ink. The methods are simple and produce large
HRLM and TEM sections in 24 hours, avoiding separate samples for LM and TEM studies. (Key words: High-resolution
light microscopy; Renal pathology; Serial sections; Electron
microscopy) Am J Clin Pathol 1983; 80: 441-444
RECENTLY, several authors have recommended the
use of TEM histotechnology to improve the study of
renal biopsies in the light microscope.1-2-78121314,27-29
This method produces HRLM sections not possible to
produce with current wax methods.18,29 The advantages
of HRLM are better preservation of tissues, seldom use
of special stains, no overlay of structures, accurate morphometry analysis, better utilization of the resolution
power of the LM, and potential use of the same block
for immunohistology.9'0,25 In addition, the large plastic
blocks prepared for HRLM can be sectioned serially and
can be used directly for high-quality TEM studies, avoiding separate samples for LM and TEM studies. Different
methods have been recommended for this latter purpose
Received January 20, 1983; accepted for publication March 28,
1983.
Address reprint requests to Dr. Hoffmann: Laboratory Service, Veterans Administration Medical Center, New Orleans, Louisiana 70146.
Laboratory Service, Veterans Administration Medical
Center and Department of Pathology, Louisiana State
University Medical Center, New Orleans, Louisiana
for tissues other than kidney. Reembedding of tissues
from plastic sections (epoxy) or fragments of the original
block prolongs (24-48 hours more) the processing time
for TEM studies and/or destroys the original HRLM
sections.4,5,6,17,22,26 The single or multiple mesa technics
waste and destroy part of the original block. 2 ' 6 The total
field method28 produces fragmentation and weakening
of the block, interfering with thin sectioning or further
sectioning for HRLM. Different methods also are reported in the literature for the serial mounting of plastic
sections (other than kidney) on glass slides.21,24 The coating of the blocks with tacky glue does not work well with
the epoxy blocks. Paraffin coating of the blocks is not
applicable when the sections are mounted on the slides
by heat, which is the most common procedure used for
mounting plastic sections. The high temperatures melt
the paraffin, interfering with the adherence of the sections to the slides. The individual transfer of the serial
section into glass slide wells is not recommended for
small blocks or when numerous serial sections are
needed. But this latter method appears to be simple, fast,
and applicable to large epoxy blocks of renal biopsies.
In this article, we describe adaptations and modifications of some of the above technics 1519,20,23 for instant
TEM thin sectioning of different parts of a large HRLM
block. Serial sections from the same epoxy blocks also
are obtained for routine renal pathology.
Materials, Methods and Findings
From 50 consecutive renal biopsies, the tips of both
ends of the needle biopsy specimens or representative
areas from open biopsy specimens are obtained for fluorescent microscopy. The central part of the needle
biopsies or the main part of the open biopsies are fixed
in paraformaldehyde fixative and postfixed on Os0 4 . 3
The specimen then is dehydrated and embedded in
0002-9173/83/1000/0441 $01.00 © American Society of Clinical Pathologists
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Vol. 80 • No. 4
LM AND TEM IN RENAL PATHOLOGY
443
FIG. 1 (upper). Serial HRLM sections (b) and TEM studies (J) are obtained from the same large block (a). An extra-thick section (c) is sectioned
from the original block (a); desired areas are selected and the extra-thick section trisected. Two selected fragments (d) are glued on the blank blocks
to obtain secondary blocks (e). These are immediately ready for TEM thin sectioning (J) or for additional HRLM sections.
FlG. 1A (lower, left). A HRLM photograph shows focal nephritis. B (lower, right). Two hours later, a TEM section from one of the secondary
blocks confirmed the presence of subepithelial, subendothelial, and mesangial deposits. An area from the other secondary block (not shown here)
presented normal glomeruli. (A, HRLM X950, B, TEM X8.500)
<
Spurr, medium viscosity.3 In most cases the molds used
to make the epoxy blocks are inverted beem capsules.
When the needle biopsies are too long (greater than 7
mm), these are bisected and trisected to fit into one block
or each fragment is embedded in a separate block. As
an alternative, and especially for the larger open biopsies, molding cups* (14 or 16 mm) also are used. However, the inverted beem capsules are preferred, because
the blocks produced here can be sectioned directly (for
HRLM and TEM) in a regular ultramicrotome. To obtain HRLM sections from the larger blocks (molding
cup blocks) a special microtomef is used.
Consecutive 1-3 /im sections for HRLM are obtained
and mounted serially on glass slides (Fig. 1). Slides have
10-15 wells painted with a water-repellent marker (any
nonwaxy, water-repellent marker is adequate) and contain a drop of distilled water per well. Printed slides with
wells are commercially available.}: One to three sections
may be mounted in each well. The slide, with the wells
and sections, is placed on a 90-100°C hot plate until
the sections are distended, the water has evaporated, and
the sections are firmly adherent to the glass (10-15 minutes) (Fig. 1). The mounted sections are stained with
toluidine blue and basic fuchsin." Immediately after
obtaining 1-3-^m HRLM sections, the face of the block
may be marked with adhesive tape for orientation purposes. This is optional, however, because the cutting
surface (polished surface) may be recognized with a dissecting microscope. A parallel extra-thick section (50200 urn) then is obtained from the same block by hand
with a razor blade or with a manual microtome with a
glass knife.20 To prevent cracking of the extra-thick sections, the blocks are warmed up with an infrared light20
to between 60-90°C. The thickness of the extra-thick
sections may vary (5-500 /tm), depending on the type
of work to be done. For routine renal biopsies, 50-200
Mm is adequate.
After studying the regular HRLM sections, glomeruli
or other areas are selected for TEM studies. If more than
one glomerulus (or tissue area) is needed for TEM studies and these are too far apart on the block face to be
cut in a single thin section, the extra-thick section is
placed in the top of a plastic petri dish and bisected or
trisected with a razor blade. Blade, specimen, and cut* Sorvall, Biomedical Division, Newton, Connecticut.
t Sorvall JB4, Biomedical Division, Newton, Connecticut.
X Roboz Instruments Co., Inc., Washington, D.C.
ting hand should be covered with a towel or petri dish
top at the moment of sectioning the extra-thick section,
because the fragments may pop out of the petri dish and
be lost. Double-faced adhesive tape may be used on the
bottom of the petri dish to prevent loss of the fragments
of the extra-thick section. Each fragment contains the
different glomeruli (or tissue areas) selected for TEM
studies. When the sections are thin (5-20 ^irn) and for
orientation purposes, these may be stained with toluidine blue. This is optional, however, because with appropriate lighting the glomeruli can be seen in the unstained osmicated sections. On the other hand, toluidine
blue staining does not interfere with TEM studies. When
2-3 or more glomeruli are close together, all of them
are processed for TEM in a single block. Blank (dummy)
blocks are prepared in advance with the same resin
(Spurr). Either side of the blank block is used, depending
on the size of the extra-thick section (or fragment of it)
selected for TEM studies. When the bottom of the block
(beem capsule block) is to be used, caution should be
taken to produce a flat bottom. This may be accomplished by polymerizing the block upside down in a
capped beem capsule, which is filled through the cut off
tip. Blank blocks are also commercially available.§ The
extra-thick sections or the parts selected from it now can
be glued to the blank block with cyanoacrylate glue**
or epoxy glueff (Fig. 1). A drop of the glue is placed on
a toothpick and thinly smeared on the flat surface of the
blank block. As soon as the glue becomes tacky (30-60
seconds), the selected part of the extra-thick section
(marked faced up) is placed on the glued part on the
block. Cyanoacrylate glue solidifies in one hour. The
glued block is placed in an incubator at 70°C for the
first 10-15 minutes and left at room temperature for the
remaining 40-45 minutes. The epoxy glue polymerizes
in the incubator in 15 minutes. The piece of adhesive
tape now is removed from the face of the secondary
block. The areas selected for TEM studies now are glued
firmly to the blank block and are ready for trimming
(if necessary) and thin sectioning for TEM studies (Fig.
2). These secondary blocks also may be used for additional 1-3-jum HRLM sections as needed. The unused
fragments of the extra-thick section may be saved in an
empty beem capsule. It is very important to leave very
§ Ladd, Burlington, Vermont.
** Hysol, E. F. Fullam, Inc., Schenectady, New York.
t t Devcon epoxy, Devcon Co., Danvers, Massachusetts.
HOFFMANN ET AL.
444
little glue on the surface of the blank block and to wait
until the glue is tacky before placing the extra-thick section. Too much glue or fluid glue (especially cyanoacrylates) will penetrate deep into the extra-thick section
and destroy the tissues. This is especially true with thinner sections. Polymerization of the glues for less than
the suggested times will produce detachment of the extra-thick section.
4.
5.
6.
7.
Comments
The present method takes advantage of the high quality of plastic (epoxy) embedding for i_M8'12-13,14,27'29 and
at the same time offers serial sections and immediate
TEM studies of selected areas of renal biopsies. All glomeruli studied by light microscopy may be studied by
TEM, even those mounted in the HRLM slides.22 The
methods are applicable to other areas of pathology where
serial sections and TEM studies are needed from different parts of the same HRLM block. HRLM serial
sections are obtained in 24 hours by individual transfer
of the sections into the glass slides. A technic to obtain
ribbons from epoxy blocks has been published recently21
and is apparently easily adaptable for renal biopsies.
TEM studies may be ready immediately (1-2 hours
later) from parallel extra-thick sections (Fig. 2). No waste
or fragmentation of the block occurs. The original and
secondary blocks remain intact with parts of the biopsy
ready for additional HRLM or TEM studies as needed.
An additional advantage is the increase in the number
of glomeruli routinely studied by TEM from each biopsy. Technicians become acquainted with sectioning
large plastic blocks for HRLM and TEM and secondary
blocks may be thin-sectioned without trimming. Our
average number of glomeruli examined by TEM before
this method was employed was two glomeruli per biopsy. With the present method, it is 3-4 and a maximum
of 12 glomeruli for TEM studies in a single large thin
section.
Potentially parallel sections from the same epoxy
blocks also may be used for immunohistology.91025 This
would avoid totally the current fragmentation of the
renal biopsy for light, fluorescent, and electron microscopy.
Acknowledgment. The authors are in debt to Karen M. Dunn for
her excellent secretarial work.
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