Routine Rapid Preparation of Thin Epoxy
Resin-embedded Sections of Renal Biopsies
for Light Microscopy
J. M.
XIPELL,
Department
M.B., B.S.,
R. C.
AND
GLADWIN,
of Patliology, University of Melbourne,
Heidelberg, Victoria 3084, Australia
A.A.I.S.T.
Austin
Hospital,
ABSTRACT
Xipell, J. M., and Gladwin, R. C.: Routine rapid preparation of thin epoxy
resin-embedded sections of renal biopsies for light microscopy. Am. J. Clin.
Pathol. 58: 469-474, 1972. A method using a low viscosity epoxy resin with
a short polymerization time for the rapid processing of renal needle biopsies
and the preparation of thin (0.4 pcm. to 1.0 ju,m.) sections is described. Sections
were stained with hematoxylin and eosin and with periodic acid-silver methenamine for light microscopy. The effects of formalin and of glutaraldehyde
fixation and of osmium postfixation on staining were evaluated. The staining
procedures are applicable to material processed for examination with the
electron microscope.
of epoxy resin-embedded thin
sections in the study of renal pathology has
become well recognized.2' *<1S Two major
problems have impeded the application of
these technics in routine diagnostic laboratories: the long polymerization time required by resins of high viscosity such as
Araldite and Epon (1 to 3 days), and the
difficulty of introducing traditional stains
into the resin. The latter problem is accentuated when osmium is present in the
tissues.12 Most workers using epoxy resins
have relied upon basic aniline dyes in neutral or alkaline solutions. 9 ' 14 ' 17 These do
not give the morphologic detail obtained
with hematoxylin and eosin, periodic acidSchiff and periodic acid-silver methenamine stains. Methods of introducing other
stains into the resin have been described.1'
2, i2,15 but involve tedious free-floating
technics, require prolonged staining times
of 12 hr. or longer, or give poor cytologic
T H E VALUE
Received October 20, 1971; accepted for publication December 1, 1971.
detail. Technics requiring removal of the
resin 7>10 can result in distortion of structure or are time consuming.
This paper describes the use of a low
viscosity epoxy resin that allows rapid processing and easy staining with hematoxylin
and eosin, and periodic acid-silver methenamine.
Materials and Methods
Fixation
Percutaneous renal biopsies obtained
with the Franklin modification of the VimSilverman needle were immediately transferred to buffered 10% formalin 3 (pH 7).
After a minimum period of 3 hr., the material was divided into 6 mm. lengths and
washed in phosphate buffered saline solution, pH 7.2 (8.5 Gm. NaCl, 1.07 Gm.
Na 2 HP0 4 , 0.39 Gm. N a H 2 P 0 4 (H 2 0) 2
made up to 1 liter with distilled water) for
5 min.
Stains were also tested on:
469
470
A.J.CJ.—Vol.
XIPELL AND GLADWIN
(1) cores fixed for 2 hr. in 2% glutaraldehyde (EM grade, Polysciences Inc.,
Warrington, Pa.) in phosphate buffer, pH 7.2;
(2) glutaraldehyde-fixed tissue postfixed
in 1% osmium tetroxide (J M C Precious Metal Products, Hatton Garden, London, England) in phosphate
buffer, pH 7.2, for 45 min.; and
(3) buffered 10% formalin-fixed tissues
postfixed in 1% osmium tetroxide in
phosphate buffer, pH 7.2.
58
lowed to polymerize at 60 C. for 6 hr.
For purposes of convenience, overnight polymerization is generally used.
Sectioning
The gelatin layer is removed and the
blocks are trimmed with a razor blade.
Sections are cut at 0.4 ^m. to 1.0 jam. on
a Reichert OMU 2 ultramicrotome, using
a glass knife with a water trough. 13 The
sections are floated onto clean glass slides,
affixed without the use of adhesives,4 and
dried on a hot plate at 60 C. for 30 min.
Dehydration
Material. Acetone (Univar Analytical
Reagent, Lab. Supply, Melbourne, Australia).
Method. The tissues are put into McCartney bottles, which are placed on a standard hematology rotator (30 r.p.m.).
The schedule used is as follows:
50% acetone in distilled water for 15
min.
100% acetone for 30 min.
100% acetone for 30 min.
100% acetone for 30 min.
Impregnation
and
Embedding
Material. Spurr low-viscosity embedding
media 10 (obtained from Polysciences Inc.,
Warrington, Pa.). The embedding medium
is made up gravimetrically using the standard composition recommended by Spurr. 18
Vinylcyclohexane dioxide, 10 Gin.
Diglycidyl ether of polypropyleneglycol,
6 Gm.
Nonenyl succinic anhydride, 26 Gm.
Dimethylaminoethanol, 0.4 Gm.
Method. The 100% acetone is replaced
by 50% epoxy resin in dry acetone at room
temperature for 15 min. This is then transferred to an incubator kept at 60 C. for
15 min.
The 50% epoxy resin is replaced by
100% epoxy resin at 60 C. for 30 min.
T h e specimens are embedded in a previously-warmed size 0 gelatin capsule mold
(Parke-Davis Co., Detroit, Mich.) and al-
Staining
Methods
All sections are immersed in saturated
alcoholic sodium hydroxide for 10 min. 7
Hematoxylin
and Eosin
Materials. Mayer's hematoxylin 3 (hematoxylin BDH Technical C.I. 75290).
Eosin. Aqueous eosin Y 130 (ICI), acetic
acid-precipitated (1 drop of glacial
acetic acid per 10 ml. of 1% (w/v)
eosin in distilled water).
Citrate buffer, pH 4 (71.1 ml. 0.2 M
Na 2 P0 4 , 122.9 ml. 0.1 M citric acid).
Method. Wash in three changes of absolute alcohol.
Wash in phosphate buffer (pH 7.2) for
5 min.
Wash in two changes of distilled water.
Wash in citrate buffer (pH 4) for 5 min.
Wash briefly in tap water.
Place in previously warmed Mayer's hematoxylin in a 60 C. water bath for 10
min.
Wash in running tap water.
Place in Scott's tap water substitute to
blue.
Wash in running tap water.
Place in previously warmed eosin in a
60 C. water bath for 15 min.
Wash in running tap water.
Air dry.
Mount in D.P.X. [dibutyl phthalate, 5
ml.; Lustrex polystyrene (Monsanto)
HF 77-105, 25 Gm.; xylene, 70 ml.].
October 1972
Periodic Acid-Silver
471
T H I N SECTIONS OF RENAL BIOPSIES
Methenamine
Materials. All solutions are made up in
deionized distilled water.
] % (w/v) concentrated periodic acid.
3% (w/v) hexamethylene tetramine
(Lab. Supply, Melbourne, Australia).
5% (w/v) silver nitrate (May & Baker,
Dagenham, England, or Merck, Darmstadt, West Germany).
5% Borax (5% (w/v) sodium tetraborate,
photographic grade)
0.1% (w/v) yellow gold chloride (auric
sodium chloride, NaAuCl 4 -2 H a O.
Hopkin and Williams Ltd., Chadwell
Heath, Essex, England.) Brown gold
chloride HAuCl 4 -3 H a O does not give
satisfactory results.
2% (w/v) sodium thiosulfate.
Stock silver nitrate-methenamine solution:
5 ml. 5% silver nitrate in 100 ml. 3 %
hexamethylene tetramine.
Working silver nitrate-methenamine solution (modified Grocott-Gomori):
20 ml. stock silver nitrate-methenamine solution.
20 ml. deionized distilled water.
2 ml. 5% borax.
Glassware used in preparation of the material and in the method is precleaned with
10% (w/v) concentrated nitric acid.
Method. The method is a modification
of a previously described technic used
in this laboratory with tissues embedded in Araldite. 5
Rinse thoroughly in distilled water.
Treat with 1% periodic acid for 30 min.
Rinse in four changes of distilled water
(from 4 to 8 sec. each).
Place slides in working silver methenamine solution at room temperature, then
transfer the slides in the silver solution to
a 60 C. water bath.
After approximately 40 min. the sections,
niacroscopically, appear light brown. They
are then checked microscopically at 2 to
3 min. intervals until the glomerular cap-
sule is black and the basement membrane
of the capillary loops is dark brown.
Rinse thoroughly in distilled water.
Tone in 0.1% gold chloride until the
basement membrane of the capillary
loops has changed from dark brown to
black and the background is almost
colorless.
Wash in distilled water.
Treat with 2.0% sodium thiosulfate for
3 to 5 min.
Wash thoroughly in running tap water.
Dehydrate through ethanol.
Clear in xylol.
Mount in D.P.X.
It is important not to exceed the stated
volume of working solution in the silver
bath, as a longer time is then needed to
attain the temperature necessary for impregnation and the silver solution tends to
deteriorate with reduction of complex silver ions to metallic silver before optimal
staining is obtained.
Results
The method has been used for preparation of sections stained with hematoxylin
and eosin and with periodic acid-silver
methenamine in times comparable to those
needed to prepare paraffin sections of similar tissues (Figs. 1 to 4). Tissue sections impregnated with periodic acid-silver methenamine have also been counterstained with
hematoxylin and eosin, to give excellent
results, the combined stain being analogous
to the method described by Jones 6 in paraffin-embedded tissue. However, maximal
clarity of basement membrane is obtained
without use of any counterstains (Figs. 3
and 4). The toluidine blue and Paragon
stains generally applied to epoxy resins as
steering stains by electron microscopists
are also easily obtained. Preliminary work
has shown good results with the Von Kossa
stain for calcium, but we have not been
able to produce periodic acid-Schiff or trichrome preparations of sufficient intensity
for photography.
472
XIPELL AND GLADWIN
Discussion
The ability to cut the 2 /*m. sections in
paraffin that are necessary for accurate interpretation of renal biopsies 11 requires
considerable technical skill and is time
consuming. Even when good 2 ju.m. sections
are obtained, resolution is insufficient to
define slight changes under oil immersion.
We have found that it is comparatively
easy for technicians to learn to cut 0.2 to
1.0 jam. sections in resin, and that a larger
number of sections can be produced relatively quickly. The length of core which
could be sectioned was limited to 6 mm.
by the cutting stroke of the ultramicrotome
available. Longer cores can be prepared
with other ultramicrotomes. 4
The time needed to produce resin sections and the difficulty of introducing standard stains have been important reasons
why epoxy resin technics have not been
more generally adopted. The use of Spurr
low viscosity embedding media, which has
an exceptionally rapid polymerization time,
overcomes the difficulty of long delay, and
the ease with which the stains penetrate
reduces the requirement for special treatments such as prolonged immersion in saturated alcoholic caustic to remove the resin
entirely 7 or treatment with hydrogen peroxide to solubilize the bound osmium
when this has been used.8 The former is
time consuming, whereas the latter introduces the danger of oxygen bubbles accumulating between the slide and the sec-
A.J.C.P.—Vol.
58
tion, particularly in thicker sections (0.6
to 1.0 jam.).
The use of buffered neutral formalin as
a fixative, followed by washing the sections
in phosphate buffer, does not necessitate
the use of special fixatives. Thus, a general
all-purpose fixative can be used, avoiding
extra work and confusion in a busy laboratory. However, material that had been
postfixed in 1% osmium tetroxide for electron microscopic studies has also been
tested, and equally satisfactory periodic
acid-silver methenamine and adequate hematoxylin and eosin preparations were
obtained. Glutaraldehyde fixation alone
and glutaraldehyde followed by 1% osmium tetroxide also gave satisfactory results. Modification of the procedure after
immersion in saturated alcoholic caustic
by washing the slides in absolute alcohol
and placing them in acid alcohol (0.5%
v/v cone. HC1 in 95% alcohol) for 5 min.
resulted in excellent differential eosin
staining being obtained in glutaraldehyde
and glutaraldehyde-osmium-fixed tissue.
This step is not necessary in formal in-fixed
material. As stated, due to the simplicity
of the method, formalin fixation was preferred; furthermore, the extra time required in the glutaraldehyde-osmium sequence would nullify the advantage of
rapid preparation. Our practice has been,
therefore, to take 1 to 2 mm. segments
from each end of the core for subsequent
electron microscopic studies. Alternatively,
the core may be split longitudinally.
FIG. 1 (upper, left). Glomerulus from case of resolving acute proliferative glomerulonephritis,
showing increased mesangium (M). Low viscosity epoxy resin-embedded. Hematoxylin and eosin.
X470.
FIG. 2 (upper, right). Oil immersion photomicrograph, showing detail of Figure 1. Mesangial
cells (MC), endothelial cells (EN), and epithelial cells (EP) are clearly seen. T h e normal basement membranes (BM) are well defined from adjacent cytoplasm. Low viscosity epoxy resinembedded. Hematoxylin and eosin. X 2,750.
FIG. 3 (lower, left). Portion of glomerulus in nicmbrano-proliferativc glomerulonephritis.
T h e silver stain is taken u p by capillary basement membrane and by mesangial fibrils (M) that
have invaded the capillary walls (circumferential mesangial interposition), giving a "double"
membrane effect. Low viscosity epoxy resin-embedded. Periodic acid-silver methenamine. X 800.
FIG. 4 (lower, right). Oil immersion photomicrograph of capillary walls in membranous
glomerulonephritis, showing the "spike" projections (S) from the outer surface of the basement
membrane. Low viscosity epoxy resin-embedded. Periodic acid-silver methenamine. X 2,750.
October 1972
THIN SECTIONS OF RENAL BIOPSIES
474
X1PELL AND GLADWIN
Spurr 16 originally suggested dehydration
through a graded series of ethanol concentrations, and also noted that the embedding mixture was compatible in all proportions with acetone, dioxan, hexylene glycol, isopropyl alcohol, propylene oxide, and
tertiary butyl alcohol. It has been found
possible using acetone to reduce the total
time required for dehydration to 1% hr.
Ethanol dehydration was also tried but
was associated with disruption of the tissue
sections when placed in alcoholic caustic.
Our experience with the silver method
has been contrary to the prediction of
other workers.1 Sufficient biologic material
is present in sections 0.5 /*m. in thickness
for adequate impregnation, and the method
has proved valuable in defining thickening
or irregularity of the basement membrane,
circumferential mesangial interposition,
and membranous transformation. It is important that the stock silver methenamine
solution be stored at 4 C. and kept for no
longer than 3 to 4 weeks, as beyond this
time variability and fading of impregnation develop.
Various hematoxylins were tested. They
included Harris's, Erlich's, and Weigert's
iron hematoxylin. Mayer's was found to be
simple to use. Using both Harris's and Erlich's stains, a greater tendency for structures normally eosinophilic in paraffin sections to stain basophilically and for normal
basophilia to diminish (staining reversal)
was observed. This suggests the possibility
of a pH imbalance due to interreaction of
tissue components with some constituent of
the resin formulation. We have not investigated this problem at this time. Weigert's
iron hematoxylin is a more tedious procedure and produces a brownish discoloration of the eosin.
Aqueous eosin Y is simpler to use than
the triosine method suggested by Lane and
Europa 7 and gave more differential staining in our laboratory. Phloxine, although
easy to use, also gave relatively poor differential staining.
AJ.C.P.—Vol.
5S
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