Large Block Embedding and "Pop-Off" Technique for Immunoelectron
Karen L. De Mesy-Jensen and by P. Anthony di Sant'Agnese, University
of Rochester Medical Center, Department of Pathology Box 626, Rochester,
Reprinted by permission of Karen L. de Mesy-Jensen
first appeared in Ultrastructural Pathology, 16:51-59, 1992
We have previously described a large block embedding
and reembedding "pop-off" technique
for specimens embedded in Spurr epoxy and destined for routine electron
microscopy (1). This technique has been of great help in our diagnostic
and research activities, allowing us to rapidly scan large areas
of tissue in semithin sections and to select specific areas for electron
microscopy. This is particularly useful in finding infrequent and
difficult to locate cells and tissue structures.
Recently, our focus has shifted to immunoelectron microscopy with
a particular interest in looking at neurosecretory granule contents
of human prostatic endocrine-paracrine cells as well as prostatic
exocrine secretory products. The endocrine-panocrine cells are relatively
sparse, and we have therefore adapted the large block embedding and
pop-off technique to immunoelectron microscopy using protein A-gold.
Three low-temperature acrylic resins were evaluated with this technique
under specified conditions of fixation, processing, and immunostaining.
Examples of the results are given with discussion of some of the
technical problems encountered and resolved.
Immunoelectron microscopy is a relatively new technique whose time
has arrived (2-4). The methods and techniques described here will
contribute to a significantly increased yield when immunoelectron
microscopic analysis is performed.
Materials and Methods
All tissue was fixed in 4% paraformaldehyde for approximately 12
hours and rinsed in Sorensen phosphate buffer. Tissue sections on
slides were immunostained using the strep-avidin-biotin method, and
thin sections were immunolabeled using 15-nm protein A-gold particles.
The antisera used were polyclonal serotonin, 1:500 (Immunoclear);
calcitonin, 1:30 (Dako); prostatic acid phosphotase, 1:250 (Dako);
and monoclonal chromogranin A, 1:100 (immunoclear).
Equipment needed includes: JB-4 microtome (H1500); Ralph knife maker;
glass strips 38 x 6mm (LMG-2); T-adapters (E9668); JB-4 moulding
cup embedding trays, 6 mm wide x 12 mm long (H1514); plastic block
holders (H1520) and chuck (H1522); dental wax; vectabond-treated
glass slides (Vector); and BEEM capsules, size 3 (BC3).
Resin Mixtures and Processing Schedules
Lowicryl mixture was 86.5 gm Monomer B, 13.0 gm Crosslink A, and
1.0 gm Benzoin ethyl ether (BEE) (5). LR Gold mixture was 100 ml
LR Gold and 4 ml Benzil. The processing schedule was:
1. 45 minutes each 50%, 65%, 80% methanol (MEOH)
2. 60 minutes each 95% and 100% (x3) MEOH at -20°C
3. 60 minutes each 1:1 MEOH/Lowicryl or LR Gold
4. 60-120 minutes 1:2 MEOH/Lowicryl or LR Gold
5. Overnight 100% Lowicryl or LR Gold at -20°C
6. Embedded and polymerized with UV (360 nm) at
The LR White mixture (cold cure method) was 100 ml LR White and
10 drops of accelerator. The processing schedule was:
45 minutes each 50%, 80%, 90% ethanol (ETOH) at
60 minutes each 1:1 ETOH/LR White ETOH at 20°C
60 minutes each 1:2 ETOH/LR White ETOH at 20°C
Overnight 100% LR White
Embedded and polymerized at 4°C
Large Block Embedding
The center hole of the H1520 plastic block backs is
filled with liquid dental wax and allowed to solidify. This helps
amount of oxygen present during polymerization. Before the tissue
is embedded, the liquid resin mixture(s) is gently bubbled with nitrogen
gas to displace dissolved oxygen. The embedding molds are filled
with liquid resin and labelled with paper labels (H1262025J) to identify
tissue. Tissue is placed into individual molds and pushed down completely,
and the molds are covered with wax-filled plastic block backs. The
tray of embedding molds is placed on a metal rack 5 to 6 inches from
an ultraviolet light source (360-nm wavelength); the tray must be
exposed directly to the light. Lowicryl and LR Gold are polymerized
at -20°C for 24 to 36 hours and LR White at 4°C for
The polymerized blocks are removed from the molds, and the backs
of the blocks are rinsed with 95% ethanol to get rid of any residual
liquid resin present. The blocks are dried thoroughly.
Large Block Sectioning
With a fine metal file, the surface of the plastic blocks are filed
carefully until the tissue is just exposed. It is important to file
a V shape at one end of the block; this will be the first part of
the block face cut, thereby decreasing knife stress as the entire
length of the block face is sectioned.
Serial semithin (3-um)sections are cut dry; sections are picked
up carefully from the knife edge with a fine-tipped forceps. Sections
are laid down dry in serial order on noncoated dry glass slides.
Every fourth or fifth section, in serial order (usually three sections
per slide), is laid on large water drops (LR Gold sections must be
floated on water and 1 drop of xylene added on top of each section)
on Vectabond-treated slides and allowed to spread until smooth. Slides
are then drained carefully and allowed to air dry vertically at room
temperature. Additional drying is done in a vacuum dessicator (the
electron microscope) for 3 hours or overnight.
Immunostaining and Matching of Adjacent Serial Sections
Plastic sections are immunostained with 10 times the antibody strength
routinely used for paraffin sections. Sections are rinsed carefully
between steps (because they will swell when exposed to water-based
solutions) and counterstained for 1 to 5 minutes with hematoxylin.
Adjacent well-dried serial sections are then matched to the areas
of interest. A dissecting needle is used to etch a rectangle around
the cells or tissue to be popped off. The back side of the slide
is dotted with a felt-tipped marker to identify where to place the
A size 3 BEEM capsule is filled with liquid resin until it is slightly
concave. The capsule is inverted quickly, placed over the etched/marked
area, and polymerized according to the plastic used. Lowicryl sections
pop off best with a resin mixture using half the amount of BEE. Polymerized
capsules are popped off the glass slides by dipping them quickly
in and out of a dewar containing liquid nitrogen (they must not be
frozen too long). Capsules will be easily lifted off. The pop-off
capsules are stained in 1.0% methylene blue at room temperature for
30 to 60 seconds, rinsed in distilled water, and dried. The capsules
are examined under a light microscope (condenser down) to determine
the flatness of the etched tissue section embedded in the pop-off's
surface. If the etched area is uniformly stained and appears in one
plane of focus, it is suitable for thin sectioning.
The molds are removed, and the excess block face is trimmed to a
mesa that contains only the etched area. The trimmed block face is
carefully aligned vertically and horizontally to a glass knife edge.
The glass knife is then replaced with the diamond knife (alignment
with the diamond knife will often only have to be horizontal). The
boat is filled with water, with the meniscus being extremely concave.
Sections are cut from dark gold to gold because they will spread
to silver-gold or silver after a few seconds of floating on the surface
of the water. Approximately 20 to 30 thin sections can be obtained
from a 3-um pop-off section. The sections are picked up on Formvar-coated
nickel grids and stained with the protein A-gold technique.
Results and Discussion
Morphologic preservation and retention of tissue antigenicity in
Lowicryl, LR Gold, and LR White resins was good compared to tissue
processed conventionally in small blocks for immunoelectron microscopy.
Nuclei, rough endoplasmic reticulum, mitochondria, and neurosecretory
granules were equally well preserved in Lowicryl and LR Gold. Some
cytoplasmic extraction was noted in tissue embedded in LR White.
Semithin (3-um) serial sections were easily cut from all large blocks.
The sections spread and adhered fairly well. The light microscopic
immunostaining for the various antigens before immunoelectron microscopy
resulted in excellent staining. One problem encountered, however,
was the continued adherence of the acrylic sections throughout the
entire immunostaining procedure. The acrylic resins are extremely
hydrophilic, and sections that had been well dried onto coated slides
would swell and lift off. Tissue sections spread and dried onto slides
coated with polylysine were not as reliable as those dried onto slides
that had been dipped in Vectabond, which is an etching agent. To
help maintain adherence of the sections, they were placed in a dessicator
(on the electron microscope) overnight and/or in a 37 degree C oven
for 30 minutes to ensure complete adherence.
Popping off adjacent serial sections was the
most difficult problem. Initially the etched tissue section would
lift up during the polymerization
of the pop-off capsules. There appeared to be two reasons for sections
lifting off the slide: The sections were not dried and attached sufficiently
to the surface of the glass slides; and the rate of polymerization
was too rapid, resulting in the section being "grabbed" off
the glass slide. The solution was ensuring thorough section drying
and using acrylic resin mixtures containing half the amount of catalyst
(this was especially true for Lowicryl). This slowed the rate of
polymerization and prevented lifting up of the section from the slide's
Pop-off capsules of Lowicryl and LR Gold resins polymerized well
and were easy to pop off. LR White's pop-off capsules polymerized
but shrunk considerably and were not large enough to be held firmly
in the metal chuck for thin sectioning. Perhaps this was due to the
increased amount of residual water contained in the plastic mixture.
The presence of oxygen in the atmosphere affected polymerization
because the capsules polymerized more uniformly in a nitrogen atmosphere.
Thin sectioning of pop-off blocks from all the acrylic resins was
similar to that of routine tissue blocks. Because of the hydrophilic
nature of the acrylic resins, however, it was necessary to use an
extremely concave water meniscus to prevent sections from being dragged
onto a wet block face. It was best to cut sections that were gold
because they spread to silver within a few seconds of floating in
the knife boat. There were enough sections to produce 15 to 20 nickel
grids, provided that the block face had been well aligned horizontally
and vertically to the knife edge before thin sectioning.
Initially we used uncoated grids for immunoelectron microscopy,
but we have switched to coated (Formvar) grids for increased section
stability under the electron beam. Immunogold labeling was excellent
with the standard protein A technique. Lowicryl and LR Gold were
more successfully adapted to our large block/pop-off method.
Routinely we are now using Lowicryl, but results appear to be comparable
with LR Gold. We are currently involved in an ongoing study to evaluate
the effects of various fixation regimens on morphologic preservation
and retention of antigenicity using the methods described here.
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