Processing Lung Tissue Without Water and Organic Solvents

Abstract

Infusion of melted paraffin into the mouse lung makes it possible to cut and process the dried-out lung tissue without water and organic solvents. The lung filled with paraffin is attached to the block and cut as conventional paraffin embedded samples that can be examined under the microscope. Alternatively, lung sections can be infiltrated with paraffin oil to remove the solidified paraffin from the alveoli and to clarify the tissue. This technique was applied to the detection of quinine distribution in the lung tissue. (The J Histotechnol 18:337, 1995)     

Tissue expansion of lung bronchi due to tissue processing for histology – A comparative analysis of paraffin versus frozen sections in a pig model

AimTissue shrinking due to fixation and processing is well known. However, the degree of shrinking varies significantly with the tissue type as well as the processing method and is not well studied in various tissues. In daily pathological routine workflow, histological specimens from frozen and paraffin sections are performed from the same tissue. In the present study we compared the thickness of bronchus walls obtained from paraffin and frozen sections.MethodsPig lungs were frozen in ventilated condition in liquid nitrogen and 36 bronchi were isolated after dissection. Frozen sections of 5 μm thickness were performed and the remaining tissue was fixed and embedded in paraffin after fixation in 4% formalin. Frozen and paraffin sections from the same cutting edge were analysed after haematoxylin and eosin staining by measuring the wall thickness of the bronchi using high power fields of 400-fold magnification. In each bronchus 40 measurements were implemented at different wall positions distributed over the entire wall area. Summed up, in each group 1440 wall measurements were performed in total. Statistical analysis was conducted using the Wilcoxon test and t-test as well as Pearson’s correlation coefficient with a significance level at P < 0.05.ResultsThe bronchial wall thickness was significantly (p < 0.001) smaller in frozen sections (median: 0.50 mm; min: 0.37 mm; max: 0.97 mm) compared to paraffin sections (median: 0.58 mm; min: 0.35 mm; max: 1.06 mm). The median difference between paraffin and frozen sections was 0.05 mm (min: -0.11 mm; max: 0.22 mm). The wall thickness ratio of both groups was as follows: frozen/paraffin section = 0.8609, thus yielding a difference between paraffin and frozen of 13.91%. High correlation was found between wall thickness measurements on paraffin and frozen sections (R = 0.87, p < 0.001).ConclusionsThe bronchus wall thickness in the frozen section was 14% reduced compared to the paraffin section. In routine pathology as well as in scientific studies these results are of relevance, as airway wall thickness represents a relevant marker for pathological interpretation, especially using CT image techniques.     

Unmasking Capacity of Stored Trypsin Solution. Rescue of Immunoglobulins in Formaldehyde-Paraffin Solution

Abstract

The effect of long term cold storage of a trypsin solution on its ability to restore immunoreactivity of immunoglobulins was investigated in paraffin sections of formalin-fixed lymphoid tissue immunostained by the peroxidase anti-peroxidase method. The length of exposure to the protease was kept constant, and aliquots from a single stock solution of trypsin were tested at 8, 12, 18, 25, 30, and 40 day intervals, using consecutive tissue sections. Formalin-fixed lymphoid tissues stained weakly or failed to stain unless they were treated with trypsin. Trypsin stored for up to 25 days rescued immunoglobulin immunoreactivity, but it was less effective at 30 days and even less so at 40 days. We conclude that the effective shelf-life of cold stored trypsin in solution used to restore immunoglobulin antigenicity is appraximately 3 weeks. (The J Histotechnol 10:229, 1987.)     

Nuclear Bubbling: An Overlooked Artifact

Abstract

Sporadic appearance of a nuclear artifact had hampered accurate microscopic evaluation of lymph node and bone marrow biopsies. Although troubleshooting included specimen handling, fixation and staining, all changes in methodology failed to produce consistently good quality sections. Improved morphologic detail was attained only after blocks were recut and sections were completely dried before heat-affixation. Complete drying of sections before heat-affixation was the only factor that consistently prevented the artifact. (The J Histotechnol 13:135, 1990).     

Modification of Brain Processing Techniques: Tissue Embedding in Glycol Methacrylate and Stain Modifications

Abstract

Increased interest in quantitation of the histopathological changes in a variety of neurological disorders (including neurodegenerative disease such as Alzheimer's disease) continues in an attempt to develop specific clinical-histopathological correlations. Most previous efforts at quantitation have used paraffin embedded sections of brain tissue, although plastic embedded sections have recently become a preferable alternative because they provide greatly reduced tissue shrinkage and distortion during processing, and greater clarity and improved resolution to the tissue sections. We have developed techniques for glycol methacrylate embedding and sectioning of brain tissue blocks on a standard histology laboratory microtome. In addition, we have modified routine diagnostic and investigational neurohistological stains for use in glycol methacrylate embedded brain sections, including hematoxylin and eosin, modified Bielschowsky stain, Jamarri silver technique, Einarson's Nissl stain, gallocyanin-Darrow red myelin stain, and the thioflavine-S-hematoxylin stain. The use of plastic embedded sections with appropriate stains will permit critical histopathological evaluation of nervous system tissue from patients with a variety of neurological disorders. (J Histotechnol 12:201, 1989)     

The changing landscape of scientific publishing

Abstract

Tissue microarray (TMA) technology makes it possible to perform high throughput in situ analysis of tissue samples, and has become a powerful tool in multiple fields of life sciences. However, obtaining high-quality TMA sections can be technically challenging. In this paper, two aspects of TMA construction were investigated, namely, the paraffin type of the recipient block and the tempering method to facilitate donor core integration. Nine commonly used paraffin types were evaluated, and four were identified with satisfactory performance. It was also found that, after TMA construction, 1-hour incubation at 50°C significantly improved the integration of donor cores with the recipient block. Large TMA blocks prepared with the described method were easy to section and yielded high quality TMA slides.     

The Cutting Edge: Abstracts from Current Literature

Abstract

A tape-transfer technique for reducing technical problems associated with the sectioning of large paraffin blocks is described. Ten different tapes were attached to the surface of tissue blocks containing decalcified bone, and sections were cut with a microtome. The tapes were evaluated for adhesion to the block face, ease of handling during sectioning, and removal before staining without disruption of the tissue. 3M Company tape numbers 3710–2 and 3750-G were found to be the most satisfactory. This tape-transfer technique yields excellent quality sections with greater ease, less distortion, and fewer artifacts than unsupported sections do. ( The J Histotechnol 13:207, 1990)     

A Rapid Stain for Campylobacter pylori in Gastrointestinal Tissue Sections Using Diff-Quik®

Abstract

Campylobactw pylori is a spiral bacteriurm recently recognized as a highly prevalent organism in the upper gastrointestinal tract. Because of its suspected pathogenetic role in gastritis and related disorders, histologic identification of the organism is often desirable. We describe a simple, rapid method for demonsmting the organism in paraffin sections of gatrointestinal tissue utilizing a commercially available, premixed min (Diff-Quick®) that gives results similar to more cumbersome Giemsa-type tissue stains. (The J Histotechnol 12:303, 1989)     

Paraffin Embedded Breast Carcinomas for the Immunohistochemical Study of Prognostic Factors

Abstract

There is increasing evidence that prognostic markers, including steroid receptors (estrogen and progesterone), estrogen-regulated proteins (pS2 and cathepsin-D), proliferation markers (proliferating cell nuclear antigen), and growth factors (epidermal growth factor receptor) can beuseful in the management of patients with breast cancer. However, a full panel of assays would need a large sample of biopsy tissue, and increasing public awareness of the need for early detection has resulted in a continuous decrease in size of individual biopsies. Commercial antibodies that perform on routine paraffin sections have now made it practical to perform an array of immunohistochemical assays on paraffin embedded tissue. Our experience with the routinely processed biopsies of 27 patients provides convincing evidence of the feasibility of determining some of the more widely used prognostic markers in breast cancer from a single paraffin block. (The J Histotechnol 15:315, 1992)     

Silver Labeling of Alzheimer Neurofibrillary Changes and Brain β-Amyloid

Abstract

Alzheimer neurofibrillary changes and brain β-amyloid can be stained selectively by silver impregnation on tissue sections and (in the case of neurofibrillary proteins) on sodium dodecyl sulfate-polyacrylamide gels. The stain can be applied to frozen sections of tissue from 10–150 μm thick, to similarly thick polyethylene glycol sections, or to 5–15 μm thick paraffin sections. The technique can also be applied to routinely fixed autopsy material. The technique takes advantage of the physical development of nucleation sites, thereby permitting tight control of the entire procedure. It is less expensive than immunocytochemical techniques, and facilitates processing of large numbers of sections through entire hemispheres of the human brain. (The J Histotechnol 16:335, 1993)     

Silver Acetate Autometallography: An Alternative Enhancement Technique for Immunogold-Silver Staining (IGSS) and Silver Amplification of Gold,Silver, Mercury and Zinc in Tissues

Abstract

Silver lactate autometallography in immunogold silver staining (IGSS) usually requires development in darkness to avoid excessive background staining. Our alternative method of silver enhancement of IGSS on paraffin sections from Bouin's or formalin fixed pancreas uses silver acetate in combination with hydroquinone in low pH buffer. The modification was tested with a range of antibodies in normal and diseased tissues (all routinely fixed and paraffin embedded), in acetone postfixed cryostat sections, and in semithin sections of glutaraldehyde fixed and resin embedded tissues. Silver acetate autometallography was also tested in various systems for the visualization of tissue metals like sulfides and selenides of mercury and zinc, silver, and gold. Comparisons between sections exposed to silver lactate and the silver acetate developer showed no significant difference in the number of structures stained. The degree of background staining was often lower when silver acetate was to used as the ion donor, especially with IGSS. The advantage the technique described here is that the development process can be controlled, using normal bright field light microscopy. (The J Histotechnol 11:213, 1988.)     

Diagnosis of Cytomegalovirus Infection by In Situ Hybridization

Abstract

A case of cytomegalovirus (CMV) infection of the bladder is described, with particular reference to the use of In Situ hybridization as a diagnostic aid. Morphologically, it was not possible to identify characteristic cytomegalic cells in the tissue. Immunocytochemistry using human serum from an infected patient gave equivocal staining due to cross reaction of the labeled anti-human immunoglobulin serum with tissue proteins. A commercially available monoclonal antibody produced clearer results. In Situ hybridization with biotin labeled CMV DNA was able to detect virus unambiguously in routinely prepared paraffin embedded tissue sections. The technique is based on the use of a high temperature to denature tissue DNA sequences, followed by simple immunological detection of hybridized biotin residues. We suggest that In Situ hybridization should be considered as a diagnostically useful tool in the histological investigation of viral infection. (The J Histotechnol 12:229, 1989).     

Subject Index

Abstract

Acrylic resin mixtures are now widely used as embedding media for the preparation of tissue sections. Most of these mixtures are based on 2-hydroxyethyl methacrylate (glycol methacrylate, GMA). Resin embedding preserves tissue components far better than paraffin, celloidin or frozen sections. The present review describes the basic principles and trouble shooting, in particular: the chemical and physical properties of GMA, and components used for GMA mixtures; fixation of tissues for resin embedding; methods for dehydration; microtomy; stretching on water and mounting in relation to the final dimensions of GMA sections; staining of GMA 3embedded tissue sections; and the use of GMA resins in immunohistochemistry. In addition, standard, step by step procedures for embedding tissues in GMA is included. (The J Histotechnol 19:297–311, 1996)     

Simultaneous assessment of DNA ploidy and biomarker expression in paraffin‐embedded tissue sections

Fleskens S J H M, Takes R P, Otte‐Höller I, van Doesburg L, Smeets A, Speel E‐J M, Slootweg P J & van der Laak J A W M
(2010) Histopathology 57, 14–26
Simultaneous assessment of DNA ploidy and biomarker expression in paraffin‐embedded tissue sections Aims: Aneuploidy is a potential biomarker for predicting progression of premalignancies. Ploidy assessment is mostly performed on nuclei isolated from tissue sections. Ploidy assessment in situ in tissue sections may be a large improvement, enabling selective sampling of nuclei, thus allowing the correlation between ploidy and histology. Existing ploidy analysis methods in sections suffer from limited sensitivity. The aim was to reliably assess ploidy in sections, combined with simultaneous assessment of other markers at the individual cell level. Methods and results: Ploidy was measured in 22 paraffin‐embedded oral premalignancies. The DNA stoichiometric Feulgen procedure was used on isolated nuclei, as well as fluoresence in situ hybridization analysis. In tissue sections, Feulgen was combined with immunohistochemistry for Ki67 proliferation marker, enabling distinction between cycling euploid and aneuploid cells. Aneuploidy was reliably detected in tissue sections (sensitivity 100%, specificity 92%). One section in which aneuploidy was detected was misclassified in isolated nuclei analysis. Sections were also successfully analysed using our model combined with DNA double strand break marker γ‐H2AX in fluorescence microscopy, underlining the power of biomarker evaluation on single cells in tissue sections. Conclusions: The analysis model proposed in this study enables the combined analysis of histology, genotypic and phenotypic information.     

Superheating Using Pressure Cooking: Its Use and Application in Unmasking Antigens Embedded in Methyl Methacrylate

Abstract

Unmasking of antigens in formalin fixed, paraffin embedded tissue sections for immunohistochemical staining has recently been reported using superheating with the aid of a pressure cooker. We describe the use and application of this technology to semithin sections of tissue that have been embedded in methyl methacrylate, the plastic we routinely employ for high resolution light microscopy immunohistochemical studies. In particular, we have investigated the use of superheating for the detection of bcl-2, CD3, CD79a, and Ki-67, as these antigens had previously proved more difficult to demonstrate following other pretreatment procedures. Using a protocol of superheating in 10 mM citrate buffer (pH 6.0) for 3 min, we easily localized all the antigens, with superior immunocytochemical staining to that achieved with microwave antigen retrieval. However, CD3 was best demonstrated when pretreatment with trypsin at 37°C was used in addition to pressure cooking. We have also shown that the choice and concentration of the accelerators N-N dimethylaniline or N-N tetramethylaniline employed for polymerizing the plastic affected immunocytochemical staining. (The J Histotechnol 21:231–236, 1998)     

Ulex Europaeus 1 Lectin for Demonstration of Lymphovascular Involvement in Microinvasive Carcinoma of the Cervix

Abstract

Lymphovascular invasion in microinvasive carcinoma of cervix may be an important prognostic indicator. However, a variety of tissue artifacts can make recognition of this invasion difficult in hematoxylin and eosin (H & E) sections. Ulex europaeus agglutinin I lectin (UEA I) used in an immunoperoxidasen technique as an endothelial cell marker facilitates the diagnosis. In this study, 32 cases with suspected vascular involvement were reacted with UEA I on both freshly cut paraffin sections and destained H & E slides. When blocks were available, adjacent sections were also studied with factor VIII antibody. Although there were problems with interpretation in some cases, there was good agreement between what was regarded as vascular space with routine staining and what was revealed with UEA I. Factor VIII was unhelpful. We concluded that UEA I immunohistochemistry is a useful adjunct for assessing capillary-like space involvement in microinvasive carcinoma of cervix. (The J Histotechnol 16:175, 1993)     

Unexpected Results of Trichrome Staining of Quenched Epithelial Tissue Following Delayed Fixation

Abstract

The histological effects of freezing and thawing unfixed tissue before experimental diffusion studies across the mucosal barrier were investigated in the face of claims that such treatment was of little significance. Following fixation, tissue sections were stained by periodic acid-Schiff (PAS), alcian blue (pH 1.0 & 2.5)-PAS, Masson and Mallory trichromes, acid-picro-Mallory phosphotungstic acid-hematoxylin, Martius-scarlet-blue, luxol-fast-blue, and Sudan black B. Trichrome type staining revealed a previously unrecorded artifact, particularly evident in quenched tissues. Three distinct epithelial cell types could be identified on the basis of differential dye uptake. This finding was not evident in PAS, alcian blue, or Sudan black B stained sections. We postulate that distortion of the cellular matrix occurs as a result of the freeze, thaw, diffusion, and fix sequence and that these cells illustrate the well known phenomenon of tissue density/dye molecular size differential staining by acid dyes. (The J Histotechnol 16:343, 1993)     

Environmentally Friendly Mercury-Free Hematoxylin

Abstract

A modified rapid method for preparing hematoxylin is described. The technique is based on avoidance of the heavy metals generally used in ripening procedures or other toxic and environmentally hazardous compounds. This formula results in a ready to use, high quality product that keeps well indefinitely. It can be used in almost every situation requiring hematoxylin staining, is equally good for staining plastic and paraffin embedded material as well as fresh frozen cryostat tissue sections, and can be used for cytological staining procedures. (The J Histotechnol 16:371, 1993)     

Alcian Blue Method for Demonstration of Histoplasma duboisii in Paraffin Sections

Abstract

A special staining technique for demonstrating Histoplasma duboisii in paraffin sections was developed during a study on the staining characteristics of this fungus, which acts as the causative organism for a type of fungal granuloma known as African histoplasmosis, found mostly in African countries. The method is relatively simple, reliable, and easily reproducible. It uses two common dyes, eosin and Alcian blue, that are readily available. The technique does not require any special fixatives and can be carried out on formalin fixed, parafin processed specimens. Because the method also eliminates the complicated and time-consuming special techniques previously used to demonstrate this fungus, it is more economical and time-saving. In paraffin sections, H. duboisii are demonstrated as round to ovoid sky-blue circlets in rose-pink background. (J Histotechnol 12:220, 1989).     

Editorial

Abstract

Hyaluronic acid films and paraffin sections of open wounds were stained with a 1% solution of Alcian blue 8GS at pH ranges between 4.0 and 2.5. Tissues were treated with Streptomyces hyaluronidase prior to staining. Untreated sections showed that material stained with Alcian blue 8GS at pH 4.0 was of increased intensity when compared to sections stained at pH 2.5. Hyaluronic acid films stained faintly at pH 2.5 and when treated with enzyme. Early wound tissue considered to be a rich source of hyaluronic acid stained intensely with Alcian blue 8GS at pH 4.0 and with decreased intensity at pH 2.5 and when digested with Streptomyces hyaluronidase. The use of Alcian blue 8GS at pH 4.0 is recommended for the study of connective tissues rich in hyaluronic acid material.