Large Block Embedding and "Pop-Off" Technique for Immunoelectron Microscopy"

Karen L. De Mesy-Jensen and by P. Anthony di Sant'Agnese, University of Rochester Medical Center, Department of Pathology Box 626, Rochester, NY 14642

Reprinted by permission of Karen L. de Mesy-Jensen
first appeared in Ultrastructural Pathology, 16:51-59, 1992

Introduction

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. 1. 45 minutes each 50%, 65%, 80% methanol (MEOH) at 4°C

2. 2. 60 minutes each 95% and 100% (x3) MEOH at -20°C

3. 3. 60 minutes each 1:1 MEOH/Lowicryl or LR Gold at -20°C

4. 4. 60-120 minutes 1:2 MEOH/Lowicryl or LR Gold at -20°C

5. 5. Overnight 100% Lowicryl or LR Gold at -20°C

6. 6. Embedded and polymerized with UV (360 nm) at -20°C

The LR White mixture (cold cure method) was 100 ml LR White and 10 drops of accelerator. The processing schedule was:

1. 45 minutes each 50%, 80%, 90% ethanol (ETOH) at 20°C

2. 60 minutes each 1:1 ETOH/LR White ETOH at 20°C

3. 60 minutes each 1:2 ETOH/LR White ETOH at 20°C

4. Overnight 100% LR White

5. 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 decrease the 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 several hours.

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 pop-off capsules.

Pop-Off Method

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.

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 surface.

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.

References

1. di Sant'Agnese PA, de Mesy Jensen KL. Diagnostic electron microscopy on reembedded ("popped off") areas of large Spurr epoxy sections. Ultrastruct Pathol. 1984; 6:247-253.

2. Bendayan M, Nanci A, Herbener GH, Gregoire S, Duhr MA. A review of the study of protein secretion applying the protein A gold immunocytochemical approach. Am J Anat. 1986; 175:379-400.

3. Herrera GA. Ultrastructural postembedding immunogold labeling: applications to diagnostic pathology. Ultrastruct Pathol. 1989; 13:485-499.

4. Roth J, Heitz PU. Immunolabeling with the protein A gold technique: an overview. Ultrastruct Pathol. 1989; 13:467-484.

5. Simon GT, Thomas JA, Chorneyko KA, Carlemalm E. Rapid embedding in Lowicryl K4M for immunoelectron microscopic studies. J Electron Microsc Techn. 1987; 6:317-324.