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.     

Chromogranin A as a marker of neuroendocrine histogenesis of tumours: an immunoelectron microscopic study with considerations about the influence of fixation and embedding media on immunolabelling

The ultrastructural localization of chromogranin A (Chr A) was studied in eleven neoplasias of the diffuse neuroendocrine system (3 pancreatic islet-cell tumours, 1 medullary carcinoma of the thyroid, 1 large bowel and 1 small bowel carcinoid tumours, 2 carcinoid tumours of the lung, 1 adenoma of the parathyroid gland, 2 pheochromocytomas of the adrenal gland). On account of the great influence of the technical treatment of the samples on the immunolocalization of Chr A, the effect of the following variables was studied in a case of pheochromocytoma: fixation in glutaraldehyde versus paraformaldehyde, postfixation in osmium tetroxide versus omission, embedding in epoxy resin versus acrylic resin. The method of choice for the better preservation of the antigenic character of the tissue was found to be fixation in 4% paraformaldehyde, omission of osmium postfixation and embedding in LRWhite acrylic resin; by this procedure we were able to find Chr A in the neurosecretory granules of all the studied cases, using three commercially available antibodies directed against Chr A. These findings further confirm that Chr A is a reliable marker for the study of neuroendocrine neoplasias by electron microscopy.     

Effect on Tissue Volume of Various Methods of Fixation, Dehydration, and Embedding

A new indirect method is described for following volume changes of homogeneous pieces of tissue during fixation, dehydration and embedding, and area changes during sectioning, staining and mounting. Pieces of rabbit kidney cortex were compared after fixation in Destin's, Orth's, Petrunkevitch's cupric-paranitrophenol, Bouin's, SUSA, Zenker-formol, 10% formalin in distilled water, formalin in saline, Burke's pyridine formalin, CaCOy neutralized formalin, MgCO₃-neutralized formalin, Bensley's vacuum distilled neutral formalin in distilled water, and Bensley's neutral formalin in saline; during dehydration in ethyl alcohol, dioxan, and tertiary butyl alcohol and clearing in xylol and chloroform; and after infiltration and embedding with parowax, with paraffin-nitrocellulose double embedding technic, with hot nitrocellulose, and with cold nitrocellulose. The H-ion concentration of these fixatives was followed during tissue fixation.

Altho all fixatives showed specific merences, SUSA and Bouin's gave the best general results and neutral formalin mixtures, especially pyridine-formalin, the poorest. Isotonic saline was found superior to distilled water as a formalin diluent, reducing tissue swelling during formalin fixation. Reagents producing marked decreases in tissue volume render such tissues less susceptible to shrinkage during subsequent procedures. Shrinkage of tissue during dehydration and infiltration with hot parffin may exceed that produced by fixation alone. Excessive heat causes tissue distortion and shrinkage. Inijltration with hot paran causes considerable shrinkage, with hot nitrocellulose Iess, and with cold nitrocellulose the least sbrinkage.     

Stereoscopic observation of enzyme distribution in lowicryl K4M-embedded specimens by conventional electron microscopy

The present investigation was undertaken to explore the value of the Lowicryl K4M embedding technique for enzyme histochemical examination of semi-thin sections. The low-temperature embedding procedure with Lowicryl K4M was found to provide favorable conditions for preservation of enzyme activity in tissue samples. We tested the histological effects of various fixatives; the best results were obtained using 4% paraformaldehyde when testing for AcPase, AlPase, TPPase, and Mg-ATPase in the dorsal root ganglion. The three-dimensional cellular fine structure could be clearly seen in stereo pair pictures under stereoscopy.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Preparation of Permanent Whole Mounts of Marine Centric Diatoms to Permit Observations of Cytoplasmic Inclusions

Marine centric diatoms are highly vacuolated plant cells which often exhibit a marked tendency to plasmolyze during fixation or other manipulatory stages required to make permanent preparations which pemit the observation of cytoplasmic inclusions. Altmann's, Schaudinn's, vom Rath's, 5% acrolein, and Allen's PFA₃ are fixatives which have been found to induce relatively little plasmolysis in the species studied. If, following fixation, the fixative is removed and desalting and dehydration are carried out in a filter assembly which allows gentle suction to make gradual changes in reagent concentration and if the cells are then placed in dilute mounting medium, only a relatively small percentage of them will be plasmolyzed. In such preparations, depending upon the fixatives employed, various cytoplasmic organelles or other inclusions will be visible in the unstained material when examined with phase contrast. Fixation with Altmann's fixative and mounting the material in Zeiss phase-contrast mounting medium (L-15) after gradual desalting and dehydration gave the best results. Standard cytochemical techniques for the detection of saccharides, protein, and lipids may be incorporated in this procedure; or, all of the steps needed for embedding the material in resins, excepting polymerization, are feasible also.     

Enzyme histochemical demonstration of NADH dehydrogenase on resin-embedded tissue

We describe a method for enzyme histochemical demonstration of NADH dehydrogenase in cold (4 degrees C)-processed resin-embedded tissue. The effects on NADH dehydrogenase activity of processing tissue through a variety of dehydrating agents and embedding in three different acrylic resins were evaluated. The optimal procedure to maintain NADH dehydrogenase activity used a short (3-hr) fixation in 1% paraformaldehyde solution, followed by dehydration in acetone and embedding in glycol methacrylate resin. Embedding of tissue in resin combined preservation and accurate localization of NADH dehydrogenase activity with good tissue morphology. Blocks of the resin-embedded tissue could be stored at room temperature for at least 6 months without loss of NADH dehydrogenase activity.     

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.     

Immunolocalization of collagen types II and III in single fibrils of human articular cartilage.

Type II and III fibrillar collagens were localized by immunogold electron microscopy in resin sections of human femoral articular cartilage taken from the upper radial zone in specimens from patients with osteoarthritis. Tissue samples stabilized by high-pressure cryofixation were processed by freeze-substitution, either in acetone containing osmium or in methanol without chemical fixatives, before embedding in epoxy or Lowicryl resin, respectively. Ultrastructural preservation was superior with osmium-acetone, although it was not possible to localize collagens by this method. In contrast, in tissue prepared by low-temperature methods without chemical fixation, collagens were successfully localized with mono- or polyclonal antibodies to the helical (Types II and III) and amino-propeptide (Type III procollagen) domains of the molecule. Dual localization using secondary antibodies labeled with 5- or 10-nm gold particles demonstrated the presence of Types II and III collagen associated within single periodic banded fibrils. Collagen fibrils in articular cartilage are understood to be heteropolymers mainly of Types II, IX, and XI collagen. Our observations provide further evidence for the complexity of these assemblies, with the potential for interactions between at least 11 distinct collagen types as well as several noncollagenous components of the extracellular matrix.     

Antigen retrieval of basement membrane proteins from archival eye tissues.

Antigen retrieval (AR) methods can unmask tissue antigens that have been altered by fixation, processing, storage, or resin interactions. This is particularly important in the study of archival tissues, because primary fixatives and storage times may vary among specimens. We performed an electron microscopic study of basement membrane components of the aqueous humor drainage pathways from archival eye tissue. AR (heated citrate buffer, pH 6.0, LR White resin) increased the amount of label of collagen IV and fibronectin in tissue fixed in four different fixatives, including those containing glutaraldehyde. Labeling density was approximately doubled after AR for most fixatives, with the largest increase for tissues fixed in 4% paraformaldehyde/2% glutaraldehyde. Duration of storage time for archival tissues did not affect AR results. AR did not change the components of the extracellular matrix labeled; no "new" components were labeled after AR. We conclude that AR in citrate buffer can be used on selected extracellular matrix antigens to enhance label that would otherwise be lost due to fixation and storage.     

Simultaneous Demonstration of Lipids and Starch in Plant Tissues

A rapid and easy technique for the simultaneous demonstration of lipids and starch in the same histological section is described. Tissues are prepared by the clamical fixing and Araldite in embedding techniques of election microscopy. Semithin sections are directly stained for 1 hour at 60C with saturated Sudan black B in 70% ethanol without removing the embedding resin. Lipids stain black; stain is shown as white grains contrasting with the blue-grey embedding resin.     

A new version of the Ag?NOR technique. A combination with DAPI staining

Summary The Ag−NOR staining technique is widely used for visualizing nucleolar organizer regions (NORs) in various plant and animal tissues. We describe a simple and time-saving combination of Ag−NOR staining with DNA detection by fluorescence microscopy. This modification was tested on cultured cells and semi-thin sections of plastic-embedded tissues. Of the different fixatives and embedding media used in our studies, the best results (i.e., high selectivity of staining, and lack of or very low background precipitation) were obtained with fixation in methanol-acetone at −20°C for cultured cells, and fixation in 4% formaldehyde followed by embedding in Histocryl resin for tissue sections. The optimal time of Ag−NOR staining was determind experimentally for all materials tested. The specificity of the staining was checked at the electron microscopical level. Especially good results were obtained by mixing epifluorescence with standard bright-field illumination. In such a combination, Ag−NOR-positive nucleoli, or their fibrillar centres and dense fibrillar components, were clearly visible against a bright background of nuclear DNA.     

Detection of albumin mRNAs in rat liver by in situ hybridization: usefulness of paraffin embedding and comparison of various fixation procedures

Our aim was to define optimal conditions for efficient and reproducible albumin mRNA detection in rat liver by in situ hybridization. We used an albumin-specific [3H]-labeled cDNA probe with a specific activity of 6-8.10(6) cpm/microgram DNA. In situ hybridization is as efficient on paraffin sections as on cryostat sections for detecting albumin mRNAs. Perfusion fixation with a 4% paraformaldehyde solution results in homogeneous RNA retention within tissue blocks, in contrast with immersion fixation, which yields heterogeneous RNA preservation. Comparison of immersion fixation with three different fixatives (paraformaldehyde, ethanol-acetic acid, and Bouin's fixative) shows that the highest level of hybridization signal is obtained with paraformaldehyde. Ethanol-acetic acid and Bouin's fixative appear less efficient for albumin mRNA detection. Loss of mRNAs within liver tissue blocks over time is largely although not completely prevented by paraffin embedding.     

The effect of fixation on the chemical extraction of glycogen from rat liver

Synopsis Small samples of rat liver, weighing 15 mg or less, were either (a) frozen in liquid nitrogen or (b) fixed at 4°C for 5 min to 2 hr in absolute alcohol, alcoholic picric acid (Rossman's fluid), or aqueous picric acid (Bouin's fluid). The tissue samples were analysed for total glycogen content by a modification of the procedure described by Goodet al. (1933).Comparable yields of glycogen were extracted from freshly frozen and fixed tissue samples. The time of fixation had no apparent effect on the amount of glycogen that could be extracted chemically. Dissolved glycogen was not detectable in the fixatives.It is concluded that (a) the fixatives used in this study do not significantly affect the yield of chemically extractable glycogen from liver; (b) fixation is extremely rapid; and (c) alcoholic fixatives are not significantly superior to aqueous picric acid fixatives for preservation of chemically extractable glycogen in very small samples of tissue.     

Enzyme histochemical investigation of glycol methacrylate embedded chick embryonic tissue

Synopsis The advantages of the water-soluble glycol methacrylate (GMA) embedding procedure make it highly applicable for use with fragile early embryonic material. Not only can one obtain tissue sections containing excellent histological detail, but numerous enzymes are retained for subsequent histochemical localization. For the purpose of establishing a methodology whereby concomitant histology and histochemistry could be obtainable, various fixatives and fixation times have been evaluated on GMA embedded chick embryonic mesonephros and gonad. It was found that fixing the tissues for 1 h in a solution of 95% ethanol, 5% acetic acid and 10% neutralbuffered formalin resulted in the retention of not only excellent histology but also alkaline and acid phosphatase. Thus, with this procedure, more specific investigations of early embryonic tissue can be performed.     

Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.     

Electron microscopy of dry peanut (Arachis hypogaea L.) seeds crushed for oil removal. Fixation and embedding of anhydrously prepared tissues

Summary Aqueous fixatives caused dry seed tissues to swell; mashed peanuts, crushed to remove oil, swelled even more. Use of anhydrous, organic solvents as vehicles for fixatives enabled maintenance of dimensional stability during fixation of dry seed tissues; even crushed seed tissue did not swell significantly when processed anhydrously. However, anhydrously processed specimens proved difficult to section. The difficulty was due to imperfect permeation of plastic into the seed tissues during embedding. An explanation of why anhydrously processed dry seed tissues are so difficult to embed in plastic is offered.     

Lymphocyte membrane antigens in glycol methacrylate embedded tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.     

Lymphocyte Membrane Antigens in Glycol Methacrylate Embedded Tissue

The use of formalin or Michel's solution either alone or in combination with acetone, and acetone, methanol or ethanol alone as fixatives, and glycol methacrylate as embedding medium were evaluated for their suitability in procedures to detect lymphocyte membrane antigens by OKT and Leu monoclonal antibodies in human tonsils. No staining was detected in sections fixed in 70% or absolute ethanol and embedded in glycol methacrylate with either the direct immunofluorescence or avidin-biotin methods. Fixation in Michel's solutions plus acetone at room temperature revealed staining by both. Neither method resulted in staining after fixation in Michel's solution plus acetone at 4 C presumably due to the slow action of the fixative. Staining was enhanced using a combination of primary and secondary biotinylated antibodies. Dual staining allowed concurrent detection of two antigens in the same section. Glycol methacrylate embedding is a possible replacement for ultracold storage in the preservation of tissue for immunofluorescent staining.