Light and electron microscopical postembedding lectin histochemistry for WGA-binding sites in the renal cortex of the mouse embedded in polyhydroxy aromatic resins LR-White and LR-Gold

Summary This work describes a technique which permits study of the postembedding lectin histochemistry for WGA-binding sites at the light and electron microscopical level on the same resin embedded tissue without removing or etching of the resin. Unfixed kidney pieces or kidney pieces fixed in 4% formaldehyde were embedded in the hydrophilic polyhydroxy aromatic resins LR-Gold and LR-White, following dehydration in up to 70% ethanol, 90% ethanol or 100% ethanol. LR-Gold was cryopolymerised at –25° C using the light sensitive initiator benzil, whereas LR-White was heat-cured at +50° C. The localisation of WGA-binding sites at the light microscopical level was investigated using FITC-labelled WGA. The ultrastructural localisation of WGA-binding sites was performed using 15 nm gold-labelled WGA. The best fluorescence staining results were obtained on fixed or unfixed tissue dehydrated in up to 70% ethanol and embedded in LR-Gold. At the ultrastructural level, the best staining results for WGA-binding sites were seen on tissue sections, dehydrated in up to 90% ethanol prior to embedding in LR-Gold.     

LR-White and LR-Gold resins for postembedding immunofluorescence staining of laminin in mouse kidney

Summary This work describes a method for the immunolocalization of laminin on 1m-thick tissue sections using a postembedding immunofluorescence technique. Embedding of unfixed or formaldehyde-fixed mouse renal cortex in either of the acrylic resins LR-White or LR-Gold permitted reliable postembedding immunofluorescence staining for laminin. LR-White was heat-cured at 50°C whereas LR-Gold was polymerized at –25°C. A stronger immunostaining for laminin was obtained from tissue embedded in polymerized LR-Gold compared with the staining from tissue embedded in LR-White. Prerequisites for adequate postembedding immunostaining are the partial dehydration of the tissue (maximum ethanol concentration, 70%) and pepsin treatment of the tissue sections prior to performing the immunostaining reactions.     

Postembedding immunogold histochemistry for the localization of laminin and its E4 and P1 fragments in mouse kidney embedded in LR-White and LR-Gold

Summary A method is presented which permits the ultrastructural localization of laminin and its E4 and P1 subunits in the renal cortex of the mouse embedded in LR-White or LR-Gold. It was performed with postembedding immunogold histochemistry using polyclonal antibodies against either the entire laminin molecular or the E4 fragment or with a monoclonal antibody against the P1 fragment. Localization of laminin was achieved in LR-White and in LR-Gold embedded kidney. Using polyclonal antibodies against the entire laminin molecule, laminin could be localized with direct as well as with indirect immunogold histochemistry with a gold labelled IgG as secondary antibody. In contrast, immunostaining for the E4 or the P1 fragments was possible only with antibodibodies directly labelled with gold.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Localization of fucosyl moieties in the mouse renal cortex by lectin histochemistry using the fucose binding lectins LTA and UEA I and by autoradiography using 3H-labelled fucose

The binding patterns of the two fucose binding lectins, Lotus tetragonolobus (LTA) and Ulex europeus I (UEA I) were investigated using fluorescence lectin histochemistry on the unfixed renal cortex of the mouse (NMRI) embedded in LR-Gold. The fluorescence staining results were compared with the autoradiographic localization of the incorporation of radioactive fucose into the renal cortex. For this study the turnover of incorporated 3H-fucose in the renal cortex was investigated 30 min, 2 h and 8 h after application. The localization of the radioactive fucose within the renal cortex corresponded well to the labelling pattern observed for lecting histochemistry using LTA. In contrast, with UEA I, no binding sites for this lectin could be observed. The results of our investigation clearly showed that fucosyl moieties in the renal cortex of the NMRI mouse are recognized by the fucose binding lecting LTA, but not by UEA I and that postembedding fluorescence histochemistry with LTA on the LR-Gold embedded kidney is a suitable technique for the localization of fucosyl moieties at the light microscopical level.     

Ultrastructural localization of WGA, RCA I, LFA and SBA binding sites in the seven-day-old mouse embryo

In the present investigation we localized binding sites for the lectins WGA (wheat germ agglutinin), RCA I (Ricinus communis agglutinin), LFA (Limax flavus agglutinin) and SBA (soya bean agglutinin) in the 7-day-old mouse embryo at the ultrastructural level. Lectin binding sites were localized on formaldehyde fixed embryos, embedded in LR-Gold, using gold-labelled lectins. Binding sites for WGA and RCA I were observed at the surface of the embryonic ectoderm oriented towards the proamnion cavity and the outer surface of the extraembryonic and the embryonic endoderm. Staining for SBA and LFA binding sites was seen in the basement membrane of the ectoderm. Moreover, binding sites for LFA were observed in the nucleoli of cells of the ectodermal, the mesodermal and the endodermal layer and in free ribosomes located in the cytoplasm of these cells.     

Localization of fucosyl moieties in the mouse renal cortex by lectin histochemistry using the fucose binding lectins LTA and UEA I and by autoradiography using 3H-labelled fucose

Summary The binding patterns of the two fucose binding lectins, Lotus tetragonolobus (LTA) and Ulex europeus I (UEA I) were investigated using fluorescence lectin histochemistry on the unfixed renal cortex of the mouse (NMRI) embedded in LR-Gold. The fluorescence staining results were compared with the autoradiographic localization of the incorporation of radioactive fucose into the renal cortex. For this study the turnover of incorporated ³H-fucose in the renal cortex was investigated 30 min, 2 h and 8 h after application. The localization of the radioactive fucose within the renal cortex corresponded well to the labelling pattern observed for lecting histochemistry using LTA. In contrast, with UEA I, no binding sites for this lectin could be observed. The results of our investigation clearly showed that fucosyl moieties in the renal cortex of the NMRI mouse are recognized by the fucose binding lecting LTA, but not by UEA I and that postembedding fluorescence histochemistry with LTA on the LR-Gold embedded kidney is a suitable technique for the localization of fucosyl moieties at the light microscopical level.     

Fixative-Dependent Increase in Immunogold Labeling following Antigen Retrieval on Acrylic and Epoxy Sections

We examined the increase in immunogold labeling of variably fixed, resin embedded tissue sections following antigen retrieval by heating in citrate solution. Fibrin clots and porcine renal tissue were fixed in glutaraldehyde, paraformaldehyde or ethanol, and specimens were embedded in LR-White or epoxy resin. Immunogold labeling was performed on ultra-thin sections with anti-fibrinogen for the fibrin clots and anti-IgG for the porcine renal tissue. Immunogold labeling increased greatly after heating epoxy sections regardless of the fixative used. The ratio labeling_retrieved/labeling_nonretrieved (L_r/L_n) was 2.8 or higher, and the largest increases were obtained for anti-IgG. Heating induced a large increase of immunolabeling for LR-White sections only when the specimens had been fixed in paraformaldehyde (L_r/L_n = 2.2 for anti-IgG and 1.4 for antifibrinogen). LR-White sections showed decreased, insignificant or weakly increased immunolabeling of ethanol or glutaraldehyde fixed tissues following antigen retrieval. Disruption of aldehyde cross-links is not the only mechanism for antigen retrieval when epoxy sections are heated in citrate solution since large increases in immunolabeling were obtained on ethanol fixed tissue. The large heat-induced increases in immunolabeling on epoxy sections are probably caused by the disruption of chemical bonds between the epoxy resin and side groups of proteins.     

Cytochemical analysis of oligosaccharide processing in frog photoreceptors

Summary Lectin cytochemistry, together with exoglycosidase enzyme digestion, has been used to characterize partially glycoconjugates of several intracellular compartments in frog photoreceptors. In order to obtain uniform access of reagents to all intracellular compartments, the experiments were performed directly on semi-thin sections ofXenopus laevis retinal tissue embedded in a hydrophilic plastic resin. In the rod, the major photoreceptor intracellular binding sites for wheat germ agglutinin (WGA) are the outer segment, the Golgi complex, and other inner segment organelles which are probably involved in the transport of glycoconjugates from the Golgi complex to the outer segment. In addition, shed outer segment tips (phagosomes) are uniformly labelled with WGA. The WGA-binding sites of the outer segment and of the presumed transport organelles are resistant to neuraminidase digestion. This is consistent with the possibility that glycoconjugates (primarily opsin) are transported from the Golgi complex to the outer segment without further oligosaccharide processing. Specific staining of rod outer segments and of phagosomes is also obtained with theN-acetylglucosamine-specific lectin, succinyl-WGA (S-WGA). Outer segments and phagosomes stain the same with WGA, S-WGA and a variety of other lectins tested suggesting that no major post-Golgi oligosaccharide processing accompanies the shedding-phagocytosis event. Concanavalin A (Con A) staining of intracellular sites in rod inner segments reveals a striking difference compared to WGA staining in that the Con A binding sites are concentrated in the photoreceptor axon and presynaptic terminal. These results, and results from previous studies, indicate that the photoreceptor may utilize different mechanisms of oligosaccharide processing from the level of a single Golgi complex to the opposite ends of this cell. Furthermore, those glycoconjugates destined for the presynaptic terminal may undergo post-Golgi processing at or near their sites of insertion into the presynaptic plasma membrane.     

Histological preparation of implanted biomaterials for light microscopic evaluation of the implant-tissue interaction

A technique is presented for processing implanted biomaterials with surrounding soft tissue for histological assessment of the implant-tissue interaction. Specimens are removed with the implant-tissue interface intact, fixed in formalin, dehydrated in a graded series of ethanol followed by a graded series of acetone in ethanol, and embedded in Spurr's low viscosity epoxy resin. Sections 0.5-1.0 mm thick are cut from the cured blocks using a metallurigical saw with a diamond wafer blade. After being glued to glass microscope slides, they are ground and polished to approximately 75 microns in thickness. The polished sections are treated with 95% ethanol saturated with sodium hydroxide, stained with Gill's hematoxylin and counterstained in eosin Y-phloxine B. The sodium hydroxide solution degrades the resin, allowing the stain to penetrate the tissue. By limiting the time in sodium hydroxide, the depth of staining is controlled and one is able to simulate a thin paraffin section with high resolution of the implant-soft tissue interface.     

Histological Preparation of Implanted Biomaterials for Light Microscopic Evaluation of the Implant-Tissue Interaction

A technique is presented for processing implanted biomaterials with surrounding soft tissue for histological assessment of the implant-tissue interaction. Specimens are removed with the implant-tissue interface intact, fixed in formalin, dehydrated in a graded series of ethanol followed by a graded series of acetone in ethanol, and embedded in Spurr's low viscosity epoxy resin. Sections 0.5-1.0 mm thick are cut from the cured blocks using a metallurigical saw with a diamond wafer blade. After being glued to glass microscope slides, they are ground and polished to approximately 75 µm in thickness. The polished sections are treated with 95% ethanol saturated with sodium hydroxide, stained with Gill's hematoxylin and counterstained in eosin Y-phloxine B. The sodium hydroxide solution degrades the resin, allowing the stain to penetrate the tissue. By limiting the time in sodium hydroxide, the depth of staining is controlled and one is able to simulate a thin paraffin section with high resolution of the imnlant—soft tissue interface.     

A method for preparing and staining histological sections containing titanium implants for light microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 microns thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

Distribution of lectin receptors sites in the zona pellucida of follicular and ovulated rat oocytes.

Carbohydrates of the zona pellucida (ZP) in mammals are believed to have a role in sperm-egg interaction. We have characterized the biochemical nature and distribution of the carbohydrate residues of rat ZP at the light (LM) and electron microscope (EM) levels, using lectins as probes. Immature female rats were induced to superovulate and cumulus-oocyte complexes were isolated from the oviduct, fixed with glutaraldehyde, and embedded in araldite for LM and LR-Gold for EM histochemistry. For examination of follicular oocytes, rat ovaries were fixed with glutaraldehyde and embedded in paraffin. The araldite or paraffin sections were deresined or deparaffinized, respectively, labeled with biotin-tagged lectins as probes, and avidin-biotin-peroxidase complex as visualant. For EM examination, thin LR-Gold sections were labeled with RCA-I colloidal gold complex (RCA/G) and stained with uranyl acetate. LM analyses indicate that in ovulated oocytes the ZP intensely binds peanut agglutinin (PNA); succinylated wheat germ agglutinin, (S-WGA), Griffonia simplisifolia agglutinin-I (GS-I) and soybean agglutinin (SBA), and to a lesser extent, lectins from Ricinus communis (RCA-I), Concanavaia ensiformis (Con A), Ulex europoeus (UEA-I), and wheat germ agglutinin (WGA). The neighboring cumulus cells are considerably less reactive and exhibit membrane staining only with Con A, WGA, and PNA. EM analysis of RCA/G binding revealed intensive binding to the inner layer region of the ZP and moderate binding to cytoplasmic vesicles of the cumulus cells. The ZP of follicular oocytes exhibits a different lectin binding pattern, expressed in staining strongly with PNA and S-WGA, and in a tendency of the lectin receptors to occur in the outer portion of the ZP.(ABSTRACT TRUNCATED AT 250 WORDS)     

A Method for Preparing and Staining Histological Sections Containing Titanium Implants for Light Microscopy

An improved method for preparing and staining ground tissue-implant sections for light microscopy is presented. Undecalcified tissue blocks with titanium implants were dehydrated in an ascending series of ethanol and stained in toto with basic fuchsin. Specimens were infiltrated and embedded in methyl methacrylate and sections were prepared using a cutting-grinding-system. The polished surface was counterstained with light green or anilin blue. Light polymerizing resin was used as slide mounting medium and for mounting the coverglass. The sections obtained were 10-15 μm thick with tissue architecture which clearly differentiated structures at the tissue-implant interface. The method was very useful for computer assisted morphometric analysis.     

Advantages in the Use of Aldehyde Fuchsin with Van Gieson's Picro-Fuchsin Counterstaining for Differentiating Bone and Cartilage

Specimens of bone were fixed in 10% neutral phosphate-buffered formalin or in Bouin's fluid and decalcified in 10% formic acid buffered with 10% sodium citrate. Materials were embedded in paraffin and 4-5 μ sections attached to slides were oxidized with 0.5% KMnO₄, decolorized in 1% oxalic acid, stained with aldehyde fuchsin, and counter-stained with Van Gieson's picro-fuchsin. Sections were dehydrated, cleared and mounted in a synthetic resin. Microscopically, the differentiation between bone and cartilage was seen as red and purple respectively, with connective tissue red; muscle and erythrocytes, yellow; and elastic fibres purple. The areas occupied by bone, cartilage and erythrocytes could be compared, and also the depth to which cartilage extended into the ossified sites. The advantages of this staining combination are: good contrasts in colour, ease of applying the stain, and virtual self-differentiation of the staining solutions.     

Evaluation of LR white resin for histology of the undecalcified rat tibia

Histology of plastic embedded undecalcified bone represents a challenging problem to the histotechnologist. We outline here an exploration of LR White resin as a suitable medium for histologic study of undecalcified rat tibia. A procedure was developed for light microscopy of rat tibia embedded in LR White and sectioned by sawing-grinding technics. The specimens were fixed in 10% neutral buffered formalin or alcohol-acetic acid-formol, dehydrated in ethanol, defatted in chloroform followed by resin infiltration and heat-curing of embedded blocks. The procedure of dehydration, defatting, infiltration, and polymerization can be completed within 10 days. Cold curing with accelerator provided by the manufacturer did not yield superior results compared to blocks cured with heat. Thick sections were obtained using a diamond wire saw, attached to plexiform slides, then ground and polished. Surface staining with Von Kossa silver reagent or toluidine blue revealed satisfactory morphological preservation of the mineralized bone sections. Artifacts like small bubbles appeared occasionally and could not be avoided despite prolonged infiltration or cold curing of blocks. Our method is relatively simple for base-line histologic study of rat tibia. The method offers advantages such as easy adaptability, reliable stainability, contrast, and resolution of bone architecture and marrow cells. Two other embedding media, Micro-Bed resin and Unicryl, were also tested, but produced inferior results.     

RAPID CHEMICAL DEHYDRATION OF PLANT MATERIAL FOR LIGHT AND ELECTRON MICROSCOPY WITH 2,2-DIMETHOXYPROPANE AND 2,2-DIETHOXYPROPANE

A rapid and simple procedure was used for chemical dehydration of plant tissue during sample preparation for light and electron microscopy. Chemically fixed tissues were washed with distilled water and then rapidly dehydrated with either 2,2-dimethoxypropane or 2,2-diethoxypropane for 15 minutes. Light microscopic observation of paraffin-embedded tissue or tissue embedded in Spurr's plastic showed excellent preservation. Electron microscopic examination of plastic-embedded tissue showed well maintained ultrastructural morphology. The dehydration procedure was also successfully applied to plant tissue destined for examination in a scanning electron microscope.     

Adaptation of the Millon, Sudan Black B, and Periodic Acid-Schiff Technics for Block Staining of Insect Tissues

Spermatophores and reproductive systems of the beetle, Lytta nuttalli Say, fixed in Bouin's aqueous picroformol or buffered 10% neutral formol were stained in toto by the Millon, Sudan black B and periodic acid-Schiff reactions as follows. Millon: after excess fixative is removed in 70% ethanol, specimens are brought to water, stained in Millon's reagent at 60 C for 1 hr, rinsed in 2% aqueous nitric acid at 40-50 C, dehydrated rapidly, cleared, embedded and sectioned as usual. Sudan black B: specimens are taken to absolute ethanol, stained in a saturated solution of Sudan black B in absolute ethanol at room temperature for 24-48 hr, rinsed and cleared in xylene, embedded and sectioned. PAS: specimens are brought to water, oxidized in 0.5 aqueous HIO₄ at 37 C for 30 min, washed in 2 changes of water, stained in Schiif reagent at room temperature for 1 hr, rinsed in 3 changes of 0.5% aqueous potassium metabisulfite, washed in running water for 10-15 min, dehydrated, cleared, embedded and sectioned. All 3 methods produced their characteristic staining in specimens up to 3 mm thick     

Binding of wheat germ agglutinin in the matrix of rat tracheal cartilage

Summary The binding of wheat germ agglutinin (WGA) to the extracellular matrix of rat tracheal cartilage was studied at both the light and electron microscopic levels. The detection of binding sites was accomplished by a postembedment method using the direct fluorescence technique for light microscopy and the avidin—biotin bridging system for electron microscopy. Distinct fluorescence was observed in the pericellular region of chondrocytes, and this fluorescence was completely removed after treatment with 4 M guanidine hydrochloride. By electron microscopy, the reaction products as found in the pericellular region were not observed in the interterritorial collagenous matrix, confirming a similar distribution as found by fluorescence microscopy. These results show that WGA-binding sites are present in pericellular matrical substances, which are known to be rich in proteoglycan and glycosaminoglycan complexes and which exhibit similar staining with antibodies to proteoglycans or with cationic dyes. As WGA binds toN-acetyl-glucosamine andN-acetyl-neuraminic acid residues, the pericellular matrix of rat tracheal cartilage appears to consist of proteoglycan having a high concentration of these saccharides.     

Light and electron microscopical demonstration of concanavalin A and wheat-germ agglutinin binding sites by use of antibodies against the lectin or its label (peroxidase)

Three straining protocols for the ultrastructural visualization of concanavalin A (ConA) and wheat germ agglutinin (WGA) binding sites were applied to samples of nervous tissue embedded in Lowicryl K4M. The hypothalamo-neurohypophysial neurosecretory system was chosen for this investigation because it has two major neuronal populations, one secreting vasopressin, whose precursor is glycosylated, and the other secreting oxytocin whose precursor form is not glycosylated. The series of incubations of the tissue sections for the three protocols were: Protocol 1: i) non labeled ConA or WGA; ii) ConA or WGA antibody; iii) protein A-gold; Protocol 2: i) pre-prepared WGA-anti-WGA complex; ii) protein A-gold; Protocol 3: i) peroxidase-labeled ConA or WGA; ii) anti-peroxidase; iii) protein A-gold. The three methods allowed to detect fine differences in the distribution of sugar residues. This, in turn, made it possible to distinguish vasopressin granules containing precursor forms from those containing processed precursor. At the light microscopic level the three methods were successfully applied to paraffin and 1-micron methacrylate sections by using a second antibody, PAP complex and the diaminobenzidine reaction.