Rapid low molecular weight polyethylene glycol embedding protocol for immunocytochemistry

We describe an alternative polyethylene glycol (PEG) embedding procedure which utilizes PEG 200 for dehydration and PEG 600 for infiltration and embedding of perfusion-fixed rat liver. PEG 600 has a melting point of 22 degrees C, enabling infiltration of fixed tissue to be performed at room temperature. Sections (2 microM) cut in a cryostat at -20 degrees C and immobilized in agarose were readily labeled by immunoperoxidase protocols with monoclonal antibodies to hepatocyte membrane antigens. Subsequent examination by light microscopy or by electron microscopy after re-embedding in resin and ultra-thin sectioning showed excellent preservation of morphology, with minimal impairment of antigenicity.     

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.     

Processing of immunoisolated pancreatic islets: implications for histological analyses of hydrated tissue

Routine tissue processing is usually associated with histological artifacts as a consequence of shrinkage and distortion during dehydration required for embedding. With hydrated specimens such as lung, embryonic, and tissues in hydrophilic membranes, tissue processing can induce severe artifacts that interfere with adequate microscopic evaluation. Here we present a method for embedding hydrophilic alginate-polylysine microencapsulated pancreatic tissue that combines the absence of histological artifacts with a practical tissue processing method. We found that the glycol-methacrylate (GMA)-embedding method preserved the integrity of the encapsulated tissue better than snap-freezing or paraffin embedding, but the overall quality of the hydrophilic capsules remained poor Next, we modified the GMA method by introducing gradual dehydration to investigate whether the integrity of the sectioned capsules was better maintained by a more gradual pattern of water extraction. This modification resulted in well-preserved morphological details of the hydrophilic membranes, hydrogel-cell interface, and encapsulated pancreatic tissue. Subsequent routine staining gave excellent contrast between the islet tissue and hydrophilic components, which allowed adequate quantitative histological and pathological comparisons.     

Serial Sectioning Techniques for A Modified LKB Historesin

A glycol methacrylate-based plastic that is capable of producing; serial sections has been introduced by LKB. This plastic, provided in the LKB 2218-500 Historesin Embedding Kit, has been tested in our laboratory for its ribbon forming capacity. Various block sizes, concentrations of the softening agent polyethylene glycol 400 (PEG), and tissue types have been examined to determine the optimal conditions for ribbon formation. Although unmodified LKB Historesin is capable of forming ribbons, these ribbons often break. The addition of PEG to the embedding solution enhances ribbon formation. When sectioning with glass knives the best results are achieved with the addition of 0.2 ml of PEG/5.0 ml of embedding medium. A conventional AO rotary microtome can be used to produce ribbons if, in addition to the added PEG (optimal concentration 0.25-0.30 per 5 ml of embedding medium) a thin layer of dental wax is added to the upper and lower surfaces of the block. Ribbons form more easily on microtomes, such as the LKB Historange, that have a retractable specimen arm. If serial sections are to be produced it is very important that the upper and lower faces of blocks be parallel.     

Serial sectioning techniques for a modified LKB Historesin

A glycol methacrylate-based plastic that is capable of producing serial sections has been introduced by LKB. This plastic, provided in the LKB 2218-500 Historesin Embedding Kit, has been tested in our laboratory for its ribbon forming capacity. Various block sizes, concentrations of the softening agent polyethylene glycol 400 (PEG), and tissue types have been examined to determine the optimal conditions for ribbon formation. Although unmodified LKB Historesin is capable of forming ribbons, these ribbons often break. The addition of PEG to the embedding solution enhances ribbon formation. When sectioning with glass knives the best results are achieved with the addition of 0.2 ml of PEG/5.0 ml of embedding medium. A conventional AO rotary microtome can be used to produce ribbons if, in addition to the added PEG (optimal concentration 0.25-0.30 per 5 ml of embedding medium) a thin layer of dental wax is added to the upper and lower surfaces of the block. Ribbons form more easily on microtomes, such as the LKB Historange, that have a retractable specimen arm. If serial sections are to be produced it is very important that the upper and lower faces of blocks be parallel.     

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.     

Ultrastructural localization of tubulin and actin in polyethylene glycol-embedded rat seminiferous epithelium by immunogold staining

The polyethylene glycol (PEG) method for immunofluorescence localization of cytoskeletal antigens has been extended to the ultrastructural level using glutaraldehyde-fixed tissues and immunogold staining. Semithin sections of fixed tissue embedded in polyethylene glycol are divested of the PEG, exposed to purified antibodies (e.g., antiactin, antitubulin) and anti-IgG-colloidal gold. The sections may be processed by dehydration and critical-point drying, or reembedment in hydrophilic substances. Tubulin is demonstrated in the mitotic spindles of dividing spermatogonia, manchettes, axonemes and centrioles of developing spermatids, and in the Sertoli cell cytoplasm; actin localization is demonstrated in the myoid cells of the tunica propria, and smooth muscle cells of arterioles in the interstitial tissue. The results demonstrate the applicability and versatility of PEG embedding for immunocytochemistry.     

The application of polyethylene glycol (PEG) to electron microscopy

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Polyethylene Glycol Embedment for Histological Studies of Bean Seed Testa of Low Moisture Content

Light microscopic examination of the structure of seed testa of snap and semihard bean seeds with 6% and 12% moisture contents in paraffin sections was unsuccessful because of poor paraffin infiltration and subsequent separation of subjacent and palisade cell layers. We devised an alternative method using polyethylene glycol (PEG) as the embedding material. Specimens were killed and fixed in the usual manner. They were then run up through a graded series (25, 50, 75, 100%) of PEG 1000 to PEG 1450, and finally embedded in a mixture of PEG 1450 and 4000 (19:1 by weight). Transverse and longitudinal sections retained excellent morphological detail and were suitable for histological study. Sections temporarily stained with 0.025% thionin allowed good quality photomicrographs.     

Polyethylene glycol embedment for histological studies of bean seed testa of low moisture content

Light microscopic examination of the structure of seed testa of snap and semihard bean seeds with 6% and 12% moisture contents in paraffin sections was unsuccessful because of poor paraffin infiltration and subsequent separation of subjacent and palisade cell layers. We devised an alternative method using polyethylene glycol (PEG) as the embedding material. Specimens were killed and fixed in the usual manner. They were then run up through a graded series (25, 50, 75, 100%) of PEG 1000 to PEG 1450, and finally embedded in a mixture of PEG 1450 and 4000 (19:1 by weight). Transverse and longitudinal sections retained excellent morphological detail and were suitable for histological study. Sections temporarily stained with 0.025% thionin allowed good quality photomicrographs.     

Immunogold localization of TGFβ 1 protein and mRNA in human skin using a colloidal gold/digoxygenin system

Tissue preservation, and immunogold cytochemical and in-situ hybridization labelling intensities vary according to the preparatory protocols used. We wished to determine which preparative protocols produce optimal preservation, protein and mRNA labelling. Nine combinations of fixative and embedding resin were therefore studied using postembedding immunoelectron microscopy and a novel immunogold digoxygenin in situ hybridization (ISH) system, to quantitate the presence of transforming growth factor beta 1 (TGF 1) protein and message in human skin. The best preservation was observed in tissue fixed in 1% glutaraldehyde and embedded in LR White resin or low acid glycolmethacrylate resin (LA-GMA). Preservation was poor in tissue fixed with 1% glutaraldehyde and fair in 4% paraformaldeyde, when embedded in Unicryl. Ethanediol dehydration coupled with LA-GMA embedding resulted in reasonable preservation. Based on quantitative measures of the labelling density for TGF 1 protein and mRNA, immunogold labelling was adequate with 1% glutaraldehyde fixation coupled with LR White or LA-GMA resins, and also with 4% paraformaldehyde and LR White resin, but was best with ethanediol dehydration and LA-GMA embedding. ISH labelling under basal conditions was best in LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde. The ISH label in tissue fixed with 1% glutaraldehyde and embedded in LA-GMA was significantly increased by treatment with proteinase K. Overall, ethanediol dehydration was associated with a good immunoelectron microscopic (IEM) label while LA-GMA with 1% glutaraldehyde or 4% paraformaldehyde resulted in a consistently detectable ISH label. LA-GMA embedding with 1% glutaraldehyde fixation gave a good result with both IEM and ISH labelling.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

Histological investigation of organisms with hard skeletons: a case study of siliceous sponges.

Siliceous and calcareous sponges commonly are treated with acid to remove the spicules prior to embedding and cutting for histological investigations. Histology of spiculated sponge tissue represents a challenging problem in sponge histotechnology. Furthermore, fluorescence in situ hybridization (FISH), a key method for studying sponge-associated microbes, is not possible after acid treatment. For a broad range of siliceous sponge species, we developed and evaluated methods for embedding in paraffin, methylmethacrylate resins, LR White resin and cryomatrix. Different methods for cutting tissue blocks as well as mounting and staining sections also were tested. Our aim was to enable histological investigations and FISH without prior removal of the spicules. To obtain an overview of tissue and skeleton arrangement, we recommend embedding tissue blocks with LR White resin combined with en bloc staining techniques for large specimens with thick and numerous spicules, but paraffin embedding and subsequent staining for whole small specimens. For FISH on siliceous sponges, we recommend Histocryl embedding if the spicule content is high, but paraffin embedding if it is low. Classical histological techniques are used for detailed tissue examinations.     

In situ localization of cartilage extracellular matrix components by immunoelectron microscopy after cryotechnical tissue processing

Localization and distribution of proteoglycans within rat growth plate cartilage were investigated by immunoelectron microscopy. By use of a mixture of three monoclonal antibodies directed against chondroitin sulfate chains and of post-embedding staining by protein A-gold, the immunosensitivity and resolution achieved by electron microscopy within tissue processed by high-pressure freezing, freeze-substitution, and low-temperature embedding were compared with those in tissue preserved by three alternative procedures (i.e., mild chemical fixation in combination with either low-temperature embedding or conventional embedding, and high-pressure freezing and freeze-substitution followed by conventional embedding). The loss of matrix components incurred during each stage of high-pressure freezing, freeze-substitution, and low temperature embedding was also determined by measuring the loss of [35S]-proteoglycans from tissue labeled in vivo, and the results compared with previously determined estimates for tissue processed using conventional techniques. Immunosensitivity, determined as the number of gold particles per unit area, was highest in tissue processed by high-pressure freezing, freeze substitution, and low-temperature embedding. Comparable results (with a reduction of only 3-7%) were achieved within tissue preserved by mild chemical fixation followed by low-temperature embedding. In both procedures where conventional embedding was adopted, sensitivity was considerably reduced (by 51% for high-pressure freezing and freeze substitution and by 74% for mild chemical fixation). Loss of matrix components was negligible during all stages of high-pressure freezing, freeze-substitution, and low-temperature embedding. Such information, and that derived from morphological inspection of the various matrix compartments in cartilage processed by high-pressure freezing, freeze-substitution, and low-temperature embedding (J Cell Biol 98:277, 1984), together demonstrate that application of this technique results in successful immobilization of proteoglycans in situ within cartilage matrix. Although loss of proteoglycans from mildly fixed cartilage embedded under low-temperature conditions is minor, morphological examination of this tissue reveals considerable shifting of proteoglycans within matrix compartments. Hence, even though immunosensitivity may be high, resolution is poor. The beauty of the high-pressure freezing, freeze-substitution, and low-temperature embedding technique is that it combines high immunosensitivity with precise localization of matrix components at the molecular level.     

Conventional xylene and xylene-free methods for routine histopathological preparation of tissue sections

Abstract

Xylene customarily has been used as a clearing agent for routine tissue processing. Because xylene is a relatively hazardous solvent, laboratories are under pressure to seek less toxic alternatives for routine use. We prepared 30 paired soft tissue specimens for routine histopathological evaluation using conventional xylene and xylene-free methods to evaluate and compare their efficacy for fixation, processing, embedding, staining and turnaround time. All specimens were measured before and after processing. Three pathologists evaluated and scored the histological sections. Tissue shrinkage was greater when using the xylene method compared to the xylene-free method. The quality of tissue sections including tissue architecture; quality of staining; preservation of epithelial, fibrous, glandular, muscle and adipose tissue; inflammatory cells; and vascular tissue was better after using the xylene method, but differences were not statistically significant. Xylene-free method produced adequate results that nearly equaled the xylene method. Added advantages included cost effectiveness, better working atmosphere and decreased toxicity.     

STUDIES ON THE PROBLEM OF PRESERVATION OF MYELIN SHEATH ULTRASTRUCTURE: EVALUATION OF FIXATION, DEHYDRATION, AND EMBEDDING TECHNIQUES

Currently accepted methods of tissue preparation for electron microscopy result in alterations of myelinated nerve fibers. In an attempt to minimize distortion of myelin, various fixation techniques, dehydration schedules, and embedding methods have been evaluated. It was found that the major damage to myelinated nerves occurs in the embedding procedure. A technique for embedding nerve tissue using the polyester Vestopal W is described which was found to result in improved preservation of myelin.     

Alkaline and Acid Phosphatase Demonstration in Human Bone and Cartilage: Effects of Fixation Interval and Methacrylate Embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.     

Alkaline and acid phosphatase demonstration in human bone and cartilage: effects of fixation interval and methacrylate embedments

Human bone and cartilage specimens were evaluated for acid and alkaline phosphatase localization following varying fixation periods for fresh or frozen tissue. Formalin fixations of up to 183 hr were followed by embedment in methyl methacrylate; frozen tissue was examined either without fixation or following fixation for up to 1 hr and subsequent glycol or methyl methacrylate embedding. The humeral epiphysis of a young patient with osteogenic sarcoma showed optimum acid and alkaline phosphatase localization following fixation for periods up to 15 hr and embedding in methyl methacrylate. Frozen costochondral junction from a newborn with osteogenesis imperfecta type II showed optimum acid and alkaline phosphatase localization following 30 min fixation in formalin and embedding in methyl methacrylate or after 5 min fixation and embedding in glycol methacrylate.