Deplasticizing of thick Epon sections involving a new adhesive technique and staining of nervous tissue

The present communication deals with a technique developed for the selective staining of neural tissue in thick (10 micron) Epon sections. A new adhesive method was needed, because the known techniques are only applicable to 0.5-2 micron thin sections. The critical step in the procedure is the adhesion of the sections onto the slides. This is accomplished by heating the sections on top of a uniform layer of albumin glycerol on the slide followed by coating with celloidin. The results after deplasticizing and coagulation with this technique are comparable to those obtained by paraffin or frozen section techniques, but in addition have the advantage of Epoxy resin embedding e.g. the possibility of cutting undecalcified hard tissues and sections for serial reconstruction.     

Stabilization of Fluorochromes After Immunofluorescent Staining of Tissue and Embedding in Epon

Tissue blocks or sections immunofluo-nt stnined before embedding, ig., liver and kidney, can be stored for more than 3 years without demonstrable fluorescence decay. The processing steps, including poststaining dehydration by alcohols and embedding in expoxy resins, seem to stabilize the fluorochromes fluo-in isotbiocyanate (FITC) and tetramethylrhodamine isothiocyanate (TRITC) so that they fade I- during illumination. This is an advantage of the preembedding, immunofluo-nt staining technique which is combined with a lack of damage to the antigens by the plastic embedding medium.     

Autofluorescence and induced fluorescence in Epon embedded tissue sections

Summary The autofluorescence of tissue components in Epon semithin sections depends on the exciting wavelength, section thickness, and the structure under observation. The degree of autofluorescence emission of a given component affects the fluorescence reaction induced by fluorochromes, a feature which must be taken into account when fluorescence microscopy is applied to the study of Epon sections.     

Autofluorescence and induced fluorescence in Epon embedded tissue sections

The autofluorescence of tissue components in Epon semithin sections depends on the exciting wavelength, section thickness, and the structure under observation. The degree of autofluorescence emission of a given component affects the fluorescence reaction induced by fluorochromes, a feature which must be taken into account when fluorescence microscopy is applied to the study of Epon sections.     

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.     

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

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.     

Histochemistry of glycosaminoglycans in cartilage ground substance. Alcian-blue staining and lectin-binding affinities in semithin Epon sections

The critical-electrolyte-concentration staining method using Alcian blue (AB) was applied to etched semithin Epon-embedded sections. The distribution of various glycosaminoglycans (GAGs) was studied in hyaline, elastic, cellular and fibrous cartilage obtained from humans and rodents. The staining patterns in semithin sections were found to correspond to those obtained using paraffin-embedded material. Lectin histochemistry was performed on consecutive sections. The following peroxidase-labelled lectins were used: Ricinus communis A I, Arachis hypogaea, Ulex europaeus A I, Triticum vulgaris, Helix pomatia, Limax flavus, and concanavalin A. The lectin-binding capacity of cartilaginous ground substance was found to be low, as was expected on account of the few free sugar residues of GAGs. Chondroitin sulphate, the most widely distributed GAG, did not exhibit lectin staining. The lectin-binding sites (positive staining for all lectins tested except H. pomatia) observed corresponded to areas positive for keratan sulphate, as shown by AB staining in preceding or following sections. The pronounced lectin binding seen in cellular structures and the inner territorial matrix regions is considered to be due to higher concentrations of oligosaccharides involved in the metabolism of GAGs.     

Simple and rapid staining for detection of Entamoeba cysts and other protozoans with fluorochromes

Three fluorochromes were applied to stain various parasitic protozoans. By double staining with 4',6-diamidino-2-phenylindole and propidium iodide, differentiation of the nuclei from the cytoplasm can easily be achieved within several seconds. The chromatoid bodies in Entamoeba cysts were stained bright red. Plasmodium yoelii at all stages except late trophozoites and young gametocytes was easily identified. In the oocysts of Cryptosporidium sp., the nuclei and cytoplasm of the sporozoites fluoresced bluish white and red, respectively, whereas the residual body appeared blue or green. The third fluorochrome, Calcofluor white M2R, was suitable for detecting the cysts of Entamoeba spp. and Chilomastix mesnili.     

Evaluation of techniques for immunofluorescent staining of microtubules in cultured plant cells

Various modifications to the immunofluorescent labeling procedures for microtubules in plant cells have been compared using cell cultures of Vicia hajastana Grossh. Using serial section electron microscopic reconstructions as a reference, we have chosen as our standard procedure a method that maximizes both the preservation of the cytoskeleton and the proportion of cells staining, while minimizing the degree of nonspecific staining. The critical steps of the procedure include stabilization of the cytoskeleton, cell wall permeabilization, and cell extraction. To maintain structural integrity during the procedure, it is necessary to stabilize the cytoskeleton with paraformaldehyde. To facilitate antibody penetration into the cell, it was necessary that the walls be made permeable via partial enzymatic digestion. Detergent extraction of cells increased the proportion of cells staining and decreased the level of nonspecific binding of the antibodies. The procedures detailed in this article provide a good starting point for the application of immunofluorescent labeling techniques to other plant systems.     

Flow cytometric analysis of antibody producing cells using double immunofluorescent staining

Summary Double fluorescent labeling, with fluorescein isothiocyanate (FITC)-labeled F(ab)₂ specific for the heavy chain and R-phycoerythrin (R-PE)-labeled F(ab)₂ specific for the light chain, was demonstrated as a convenient means for the accurate evaluation of a heterogeneous non-antibody-producing population. Furthermore, it could be used for monitoring the changes in each immunoglobulin (Ig) chain content of the cells during the batch culture, which will facilitate the study on antibody synthesis, assembly and secretion.     

Fluorescent vital staining of plant sexual cell nuclei with DNA—specific fluorochromes and its application in gametoplast fusion

DNA－binding fluorochromes are often used for vital staining of plant cell nuclei.However,it is not always sure whether the cells after staining still remain in living state.We chose several criteria to estimate the validity of real vital staining for sexual cell nuclei.These were:the cytoplasmic streaming in pollen tubes whose nuclei were stined,the simultaneous visualization of fluorochromatic reaction and nucleus staining in isolated generative cells,and the capability of isolated.prestained generative or sperm cells to fuse with other protoplasts.The results confirmed that 4,6-diamidino-2-phenylindole(DAPI),Hoechst 33258 and mithramycin could be used as real vital stains,though their efficiency varied from case to case;among them DAPI showed best effect.The fluo rescent vital staining technique offered a useful means foridentification and selection of heterokaryons in gametoplast manipulation studies.