The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

Intensity of Feulgen staining of rat liver nuclei fixed with two different concentrations of formalin

Summary The results presented in this paper indicate that following fixation of rat liver in either 40% (w/v) or 10% formalin solution, Feulgen staining is far greater in the tissue fixed in the former fixative as compared with the same fixed in the latter. A possible mode of action of formalin towards fixation and subsequent Feulgen staining is suggested.     

Effect of fixation on immunolocalization of transferrin and albumin in the liver of the adult rat

The influence of the fixation procedure on the localization of albumin and transferrin in adult rat liver has been carried out using an indirect immunoperoxidase technique at the light and electron microscopic levels. Perfusion and immersion fixations with different concentrations of paraformaldehyde (with or without addition of glutaraldehyde) have been investigated. According to the mode of fixation (perfusion versus immersion) and the concentration of the fixative, the number of albumin and transferrin containing hepatocytes could vary from 10% to 100%, and different labeling patterns could be observed at the electron microscopic level. For the same concentration of fixative, a perfusion fixation induces a less intense labeling than an immersion fixation. Thus similar results are obtained after immersion fixation in 6% paraformaldehyde + 0.25% glutaraldehyde or after perfusion fixation in 4% paraformaldehyde + 0.025% glutaraldehyde. Similar data are noticed after immersion fixation in 4% paraformaldehyde or after perfusion fixation in 1% paraformaldehyde + 0.025% glutaraldehyde. Moreover, perfusion fixation induced a more fine cell structure preservation than immersion fixations and avoided the appearance of zones of fixation.     

A Simple Method for Histochemical Demonstration of Viral Inclusion Bodies in Cell Monolayers in Microtitration Plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.     

An improved method for preparing whole specimens from bovine preimplantation embryos: A technique note

A method was devised to prevent loss of whole embryos during fixation. Specimens were prepared in a chamber saturated with fixative vapors consisting of 3 : 1 (v/v) 96%. ethanol/glacial acetic acid. Good quality specimens were obtained after fixation for at least 24 but not more than 72 h. After staining, specimens could be preserved for 3 to 4 d by storage in the fixation chamber, in 45% aqueous acetic acid vapor. Using the method suggested in this paper prevents loss of early embryos during fixation and allows storage of specimens for longer than usual time while maintaining the quality of the specimen.     

A simple method for histochemical demonstration of viral inclusion bodies in cell monolayers in microtitration plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.     

Pronase treatment increases the staining intensity of GABA-immunoreactive structures in the paravertebral sympathetic ganglia

A novel tissue preparation technique for improving gamma-aminobutyric acid (GABA) immunocytochemistry has been developed. The influence of the glutaraldehyde concentration in the fixative and the effect of pronase treatment on the GABA immunostaining were tested. This method includes fixation with a high concentration of glutaraldehyde, gelatin embedding and treatment of the sections with pronase. In sympathetic (paravertebral) ganglia and their connectives, the most intense and specific immunoreaction was obtained with the following procedure: immersion fixation in 5% glutaraldehyde, infiltration and embedding in 15% gelatin, secondary fixation of the samples with 4% formaldehyde, floating frozen sections and digestion with 0.1% pronase for 15-20 min. With this technique, the GABA-containing structures (cells and nerve fibers with varicosities forming basket-like networks around some principal neurons) were selectively labeled. The data presented suggest that (1) a high concentration (5%) of glutaraldehyde in the primary fixative is necessary to preserve a large proportion of the GABA content; (2) this glutaraldehyde fixation partly masks the GABA immunoreactivity; and (3) this masking may be overcome by a proteolytic treatment preceding the immunostaining. This method has been extensively tested for the light microscopic visualization of GABA-containing tissue components in the sympathetic ganglion chain, but it may probably also be used for the immunocytochemical detection of other small molecules in other parts of the nervous system.     

Carbodiimide as a tissue fixative in histamine immunohistochemistry and its application in developmental neurobiology

The object of this study was to develop an immunohistochemical method that could be used to study neuronal histamine, especially in nerve fibers and terminals where most previous methods have not been applicable. Three new antisera were produced in rabbits against conjugated histamine, and the fixative used in conjugation, 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide (EDCDI), was used in tissue fixation and compared to paraformaldehyde. Specificity of the antisera was established with dot-blot tests on nitrocellulose, with blocking controls and affinity-purified antibodies. EDCDI appeared to be superior to paraformaldehyde as a fixative, and histamine-immunoreactive nerve cells were visualized in developing rat brain during late fetal development from embryonal day 12. By the second postnatal week, the distribution of histamine-immunoreactive neurons in rat brain had reached the adult pattern and immunoreactive nerve fibers were seen in many areas. Posterior hypothalamic neurons from newborn rat in vitro showed strong immunoreactivity for histamine and developed long varicose fibers, which covered the culture dish by the end of the fourth week in vitro. Fixation with EDCDI also allowed detection of histamine in gastric enterochromaffin-like cells and mast cells in rat. The results suggest that the histamine-containing neuron system in rat brain develops during the late fetal and early postnatal periods, and that immunoreactive neurons develop long fibers both in vivo and in vitro.     

The importance of the fixation of colour,pattern and form in tropical Pseudocerotidae (Platyhelminthes,Polycladida)

At last a fixation method that ensures tropical pseudocerotid polyclads are fixed flat, preserved for histological preparation and which also retains their colour pattern has been developed. FCA-PGPP (Formaldehyde Calcium Acetate-Propylene Glycol, Propylene Phenoxetol) fixative is frozen and worms are coaxed onto filter paper which is then laid on the frozen fixative. As a consequence, over 230 species have been documented from the southern Great Barrier Reef and eastern Papua New Guinea (Newman & Cannon, 1994; in press). It was determined that species diagnoses need to be based on colour pattern, general morphology of living animals and serial reconstructions of the male anatomy.Christensen Research Institute Contribution No. 82     

Fixation variables in horseradish peroxidase neurohistochemistry. I. The effect of fixation time and perfusion procedures upon enzyme activity.

In a series of neurohistochemical experiments the effect of aldehyde fixation upon the detection of horseradish peroxidase (HRP) was examined. These experiments demonstrated that: a) Increments in fixation of as little as 1 hr significantly decreased the number of labeled neurons; 12-hr fixation abolished HRP activity in many neuronal populations and significantly reduced the apparent size of the injection site. b) This negative fixation effect was greatest where the HRP concentration was low (e.g. in small, lightly labeled neurons) but was still evident in areas of high concentration (e.g. large, heavily labeled neurons). c) This effect was also most prominent when a less sensitive diaminobenzidine histochemical procedure was employed but was still apparent with a more sensitive benzidine dihydrochloride procedure. d) Immersion of the brain in fixative after perfusion produced a greater attenuation of HRP activity in more superficial areas. e) Immersion of the brain in buffer to terminate fixation produced a prolonged and unpredictable gradient of fixation. f) Excess, unbound fixative inhibited the histochemical reaction per se and had to be removed from the tissue but prolonged washing did not resurrect enzyme activity which was lost by fixation. To obviate these problems and optimize HRP enzyme activity a new perfusion-fixation procedure was developed. It entails 30 min fixation by perfusion which is terminated by a subsequent 30 min perfusion with cold sucrose-fuller to wash out unbound fixative. This allows the tissue to be processed immediately, produces a uniform and morphologically adequate fixation, and minimizes the negative effects of fixation on HRP enzyme activity.     

Development and application of a novel histological multichrome technique for clam histopathology

A novel and expedient histological tetrachrome technique was developed and applied to whole-body sections of the clam Ruditapes decussatus (L. 1758). The technique involves fixation in Carnoy's fluid followed by immediate embedding in paraffin with staining with a combination of Alcian Blue, Periodic Acid-Schiff's, Haematoxylin and Picric Acid. Fixation and staining was perfect for all tissues and resolved good identification of Perkinsus sp. infection and high structural detail. Among the surveyed fixatives, Bouin-Hollande's fluid also provided good results, however, fixation is potentially longer, polysaccharide staining was less intense and fibres appeared to be better preserved by Carnoy's.     

Pronase treatment increases the staining intensity of GABA-immunoreactive structures in the paravertebral sympathetic ganglia

Summary A novel tissue preparation technique for improving gamma-aminobutyric acid (GABA) immunocytochemistry has been developed. The influence of the glutaraldehyde concentration in the fixative and the effect of pronase treatment on the GABA immunostaining were tested. This method includes fixation with a high concentration of glutaraldehyde, gelatin embedding and treatment of the sections with pronase. In sympathetic (paravertebral) ganglia and their connectives, the most intense and specific immunoreaction was obtained with the following procedure: immersion fixation in 5% glutaraldehyde, infiltration and embedding in 15% gelatin, secondary fixation of the samples with 4% formaldehyde, floating frozen sections and digestion with 0.1% pronase for 15–20 min. With this technique, the GABA-containing structures (cells and nerve fibers with varicosities forming basket-like networks around some principal neurons) were selectively labeled. The data presented suggest that (1) a high concentration (5%) of glutaraldehyde in the primary fixative is necessary to preserve a large proportion of the GABA content; (2) this glutaraldehyde fixation partly masks the GABA immunoreactivity; and (3) this masking may be overcome by a proteolytic treatment preceding the immunostaining. This method has been extensively tested for the light microscopic visualization of GABA-containing tissue components in the sympathetic ganglion chain, but it may probably also be used for the immunocytochemical detection of other small molecules in other parts of the nervous system.     

The development of a new technology for controlled cell fixation. A methodological pilot study.

Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation (1) the fixative is always supplied in excess of the cell sample, and (2) the process of fixation is influenced by chemical impurities of the fixative fluid. Both factors influence the subsequent staining of cells. In order to avoid these uncontrolled influences, a new technology for controlled cell fixation has to be developed, whereby freshly prepared formaldehyde gas in an "inert" gas-flow of helium was applied to thin membranes by use of a capillary flow-in technique. The amount of fixative gas supplied, adsorbed, absorbed, diffused, and desorbed after saturation of the membranes could be reliably measured with an on-line operating "inert" mass spectrometer of the Omegatron type.     

Influence of fixation procedures on the microanalysis of lead-induced intranuclear inclusions in rat kidney

Using Laser Microprobe Mass Analysis (LAMMA), we studied the chemical composition of lead-induced intranuclear inclusions in rat kidney tissue prepared by three different wet chemical fixation procedures for transmission electron microscopy. Fixation with glutaraldehyde-Na2S gave the same results as fixation with glutaraldehyde only: a high lead concentration could be detected. Therefore, for lead strongly bound to proteins, precipitation procedures are not essential. Post-fixation with osmium tetroxide drastically changed the composition of the inclusions: the lead concentration decreased substantially, while sodium, calcium, and barium were introduced. The osmium tetroxide fixative was found to be the source of the contamination. It also contained aluminum, and we suggest that other proteins (e.g., in neurofibrillary tangles) might be able to take up Al out of solution and that care must be exercised in interpreting the microanalytical results of osmium-fixed material. For the microanalysis of the lead inclusions, fixation with glutaraldehyde only provides a good compromise between preservation of the ultrastructure and maintenance of the element distribution.     

Development of a rapid method for detecting bacterial cells in situ using 16S rRNA-targeted probes.

A rapid method for the identification of bacterial cells using 16S rRNA-directed, fluorescently tagged oligonucleotide probes has been developed. The parameters evaluated for their effect on labeling intensity included storage time, type of fixative, time of fixation, treatment time with methanol:formaldehyde and treatment time with borohydride. The results of tests using a variety of microorganisms, both Gram-positive and Gram-negative, are presented. Using this method, cells are spotted onto slides and stored desiccated until hybridized. This method may be especially applicable to environmental samples, which comprise diverse cell types and frequently require storage prior to examination.     

Immunocytochemical evidence for the presence of oxytocin in rat testis

Summary The presence of oxytocin, vasopressin and neurophysin in the testis of adult Wistar and Brattleboro rats has been examined immunocytochemically. After fixation in modified Bouin's solution, or Bouin's sublimate fixative, immunostaining was accomplished with the peroxidase-antiperoxidase method. The presence of immunoreactive oxytocin was demonstrated in 80% of the interstitial cell population of both rat strains while no staining was observed for vasopressin or neurophysin.     

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.     

USE OF POTASSIUM PYROANTIMONATE IN THE LOCALIZATION OF SODIUM IONS IN RAT KIDNEY TISSUE

Potassium pyroantimonate added to fixative solutions has been used in tissue localization of sodium ions. The distribution and specificity of the resulting precipitate in rat kidney is described in this study. Two reproducible patterns of precipitate were obtained in control tissues. The first pattern, which occurred after fixation in solutions containing aldehyde, showed the precipitate to be mainly extracellular. The second pattern, showing the precipitate in both intracellular and extracellular locations, occurred after aldehyde fixation in those experimental situations favoring cellular swelling or after fixation with solutions containing osmium tetroxide. It appeared that sodium ions could move after fixation but that sodium pyroantimonate precipitate could not. Since model systems demonstrated that dense precipitate formed when potassium pyroantimonate was added to solutions containing certain biological amines or some divalent cations, it appeared likely that the reagent did not provide specific tissue localization for sodium ions.     

A fixation method for improved antibody penetration in electron microscopical immunoperoxidase studies.

A fixation method for electron microscopical immunoperoxidase staining has been developed, which (a) allows penetration of antibodies through cell membranes to intracellular antigen sites, (b) provides a reasonable cell preservation and (c) does not alter the antigenic structure in too great an extent. Penetration of the antibodies has been achieved by using saponin as a cell membrane attacking agent. The best results could be obtained after pretreatment of cell monolayers with a mixture of 0.05% saponin, 0.0125%-0.05% glutaraldehyde and 1% paraformaldehyde for 5 min at 4 degrees C, and postfixing them with the corresponding fixative without saponin for 45 min at 4 degrees C.     

A methodological study for the quantification and the control of the physico-chemical processes occurring during cell fixation

Summary Fixation in a traditional sense means the immersion of biological material into a chemical fluid. For permanent preservation the fixative is always offered (1) in excess of the cell sample, and the process of fixation is influenced by (2) chemical impurities of the fixative fluid.Both factors influence the succeeding dyeing of cells. In order to avoid these uncontrolled criteria, a new technology for controlled cell fixation has been developed, whereby freshly prepared formaldehyde and methanol gas in an inert gas-flow of helium was applied to thin membranes by aid of a capillary flow-in technique.The instrumental equipment consists of (1) an ultra-high vacuum flowapparatus with a total-pressure measuring unit, (2) a gas-supply device, (3) a mass spectrometer including a pump system, and (4) a Teflon and/or glass-gas chamber for the treatment of synthetic (Hostaphan foils) or biological membranes (mesenterium) with formaldehyde as the fixative gas.The amount of offered, adsorbed, absorbed, diffused, and desorbed fixative gas could be absolutely estimated after the saturation of the membranes with an on-line operating inert mass spectrometer of the Omegatron type.The gas treatment of the Hostaphan foils with formaldehyde showed that nearly all adsorbed gas molecules could be desorbed. In contrast to native membranes the greatest proportion of the gas molecules adhered to the biological surface, and only a small quantity were desorbable. Physisorption or physisorption and chemisorption occured depending on the adsorber surface property.A monolayer of formaldehyde of 5·10¹⁴ to 1·10¹⁵ molecules per 10¹⁶ Ų surface area can be postulated on the basis of these preliminary results. This value corresponds to a mass of about 5·10^–8g CH₂O. It resulted in an area-coverage ratio of CH₂O molecules per cell of 10⁹:1.The membrane surface facing the gas side always amounted to 1 cm². A fixative gas concentration of 10⁶ molecules/cm³, and therefore a degree of coverage of <1/1000 monolayer can be estimated absolutely. For a precise determination of the degree of fixation, further experiments and the evaluation of additional physico-chemical parameters are necessary.