Microwave Fixation of Whole Fetal Specimens

This study compares microwave fixation of whole fetal specimens with conventional techniques performed at room temperature. All fetuses were obtained from the same pregnant rat; half of them were placed in neutral formalin for 15 min at room temperature, then irradiated for 2.5 min in a domestic microwave oven. The remaining fetuses were placed in neutral formalin at room temperature for 48 hr as a control. Both experimental and control groups were exposed to routine tissue processing for light microscopy and embedded in paraffin wax. Sections 5 μm thick were stained with hematoxylin and eosin. Our results showed that the microwave technique reduced the fixation time while providing thin sections that were equal to or better in quality than those in the control group.     

Immunohistochemical detection of p53 protein in basal cell skin cancer after microwave-assisted antigen retrieval

p53 is the most frequently altered tumor-suppressor gene in skin cancer. In normal tissues the p53 protein (wild type) has a very short half-life and it is not detectable immunohistochemically. In contrast, the mutant p53 protein has an extended half-life in tumor cells and can be detected by immunohistochemical methods. p53 is widely used as an indicator of tumor aggression and progression. Fixation methods especially formaldehyde based fixation may mask the immunohistochemical detection of p53 protein but antigen retrieval methods enhance the inmmunohistochemical detection of p53 protein by remodification of protein structure. This study was designed to evaluate the efficacy of different fixatives, of microwaving and microwave pretreatment method to retrieve p53 immunoreactivity in paraffin-embedded non-lesioned (adjacent normal tissue) human skin samples or pathological human skin samples diagnosed as basal cell carcinoma. The samples were fixed at RT and/or in microwave oven either in neutral buffered formalin or alcohol for different time periods. For antigen retrieval, the sections were irradiated in a microwave oven for 5 cycles in 10 mM citrate buffer (pH 6.00). In this study the effects of six different fixation methods on the immunohistochemical staining have been investigated in basal cell tumor specimens. The application of antigen retrieval method was also examined and compared. Optimal results were obtained using samples fixed in alcohol either at room temperature (24 h) or in microwave oven.     

Differential Staining with a Mixture of Safranin and Fast Green FCF

Mounted deparaffinized sections were stained for 30-60 minutes at room temperature in a mixture of equal volumes of 0.1% aqueous solutions of safranin O and fast green FCF filtered before use. They were then washed in distilled water for 5 minutes, blotted, washed in 2 changes of absolute alcohol (2-3 min) and mounted from xylene. The nucleic acids are stained purplish-red, half esters of sulfuric acid orange, and proteins green. The procedure is applicable to a variety of materials fixed in a number of reagents though best results are obtained after acetic-alcohol fixation. Bouin's fluid and 10% neutral formalin are not suitable fixatives for this procedure. After acetic-alcohol fixation, the staining procedure may be used in conjunction with enzyme or extraction technics in order to characterize certain chemical components of cells or tissues. The safranin-fast-green technic has proved useful in investigations of pathological changes in tissues; in the visualization of secretory granules and in studies of cellular differentiation. The technic also would appear to greatly facilitate mitotic index determinations.     

Rapid microwave fixation--a comparative morphometric study

Summary Tissue blocks taken from healthy human lung tisues, from primary bronchial carcinoma and from mediastinal and hilar lymph nodes were placed in the following solutions. Tris buffer; buffered formalin (0.5%, 1%, 7%); 0.1 mol NaCl; distilled water; DMSO (1%, 10%, 20%); acetone (10%); methanol (50%, 80%, 100%); glutaraldehyde (2.5%), and fixed by use of a commercial microwave oven.Tissue blocks obtained from the same surgical specimens were fixed in 7% buffered formalin for 24 h for comparison. Conventional and microwave-fixed tissue was embedded in paraffin, and stained with haematoxylin and eosin. Fifteen specimens of each group and each solution were examined by light microscopy. Minimum diameter and area of 100 nuclei of each specimen were measured interactively.Histomorphological sections fixed with Tris buffer in a microwave oven revealed best morphological results showing more contrast in chromatin distribution of nuclei and opening of interstitial lung capillaries in comparison to conventional formalin-fixed specimens. No statistically significant differences in area and minimum diameter of nuclei between the different groups were found. Microwave fixation using Tris buffer is a time-saving fixation method at least comparable to conventional formalin fixation. It is not accompanied by hazards to the environment that are unavoidable by formalin fixation.     

Application and Evaluation of the Lead-Adenosine Triphosphatase Method in Skeletal Tissue

Application and evaluation of the lead-ATPase histochemical method in skeletal tissue has demonstrated an intracellular localization of enzyme activity. The skeletal tissue was demineralized for 72 hr in cold 10% aqueous EDTA adjusted to pH 7.2. Frozen sections were cut and placed on cold albumenized slides, oriented, thawed, dried in a cool air stream, and fixed for 10 min in cold (-2 to -3 C) 10% formalin buffered with Na-acetate and adjusted to pH 7.2. The sections were washed, treated with 10% EDTA for 20 min at room temperature, rewashed, and incubated for an optimal period of 30 min at 37 C. in the lead-ATP medium of Wachstein and Meisel. Following incubation the sections were washed, treated for 1 min with 1% (NH₄)₂S, rewashed, immersed for 30 min in 10% buffered formalin, dehydrated, cleared, and mounted. Evaluation of the substrate specificity suggests that other phosphatases associated with skeletal tissue do not complicate the ATPase reaction.     

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.     

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.     

Acceleration of Mallory's Phosphotungstic Acid-Hematoxylin Staining for Skeletal Muscle Fixed in Formalin

The staining time for mammalian skeletal muscle fixed in neutral phosphate-buffered formalin was shortened from 12-24 hr to 10-30 min. The permanganate-oxalate sequence was omitted although oxidation by periodic acid or with iodine was found to be necessary. The material was embedded in paraffin and cut 6 μ or less. Deparaffinized sections were treated with 1% alcoholic iodine for 10 rain followed by 5% Na₂S₂O₃ for 2 min and placed in an oven at 60 C for 10-30 min to stain in a preheated mixture of 50 ml of ripened Mallory's phosphotungstic acid-hematoxylin and 1 ml of 2% phosphomolybdic acid. Experiments with fixation showed that the staining procedure followed Zenker's fluid successfully but not Bouin's fluid. Oxidation by KMnO₄ was effective only after Zenker fixation; oxidation by CrO₃ was unsuccessful.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

Rapid Bielschowsky silver impregnation method using microwave heating.

The Bielschowsky silver impregnation method has been used extensively to demonstrate neuronal processes including dendrites, axons and neurofibrils. In this study, we examined the differences in the time required for and the staining quality of the Bielschowsky method for neuronal processes when microwave heating was used instead of processing at room temperature. For this purpose, a control group of sections stained according to the conventional method at room temperature was compared to an experimental group stained in a microwave oven at 180 W for 2, 4 and 1 min in 2% silver nitrate, ammoniacal silver nitrate and gold chloride, respectively. Light microscopic examination demonstrated that the normal structure was preserved in both groups and that there was no difference in the staining quality between the control and the microwave groups. In addition, staining time for this procedure was reduced to 8 min by using the microwave oven. Our study revealed that microwave irradiation can be used safely for Bielschowsky silver impregnation of neuronal tissues.     

A histological comparison of phase-partition fixation with fixation in aqueous solutions

In phase-partition fixation, tissue is immersed in a non-aqueous solvent at equilibrium with an aqueous solution of a fixing agent to minimize osmotic effects. Preservation of morphology afforded by phase-partition fixation using formalin and glutaraldehyde and several organic solvents was compared to aqueous 10% neutral buffered formalin fixation for five tissues. It was shown that phase-partition fixation can provide excellent fixation for light microscopy if the proper combinations of fixatives and solvents are used.     

Detection of proliferating cell nuclear antigen in diagnostic histopathology.

We describe the effects of tissue preservation, fixation time, and hydrolytic treatment on the detection of proliferating cell nuclear antigen (PCNA) by immunoperoxidase staining with three commercial anti-PCNA antibodies (19A2, 19F4, PC10). Our goal was to provide guidelines for PCNA immunohistochemistry in formalin-fixed, paraffin-embedded specimens. In proliferative cell compartments, nuclear staining was achieved with all three antibodies. In some cases PCNA was also expressed in non-proliferative, histologically normal tissues associated with tumors or other lesions elsewhere. In most autopsy specimens PNCA immunoreactivity was markedly diminished as compared with similar surgical specimens. Incubation overnight with primary antibody at 4 degrees C enhanced PCNA immunoreactivity over incubation at 42 degrees C for 45 min. Pre-treatment with 2 N HCl did not increase staining. Staining with the PC10 antibody was much better preserved than staining with the antibodies 19A2 and 19F4 after prolonged formalin fixation of surgical specimens and in tissues obtained at autopsy. With all three antibodies, however, PCNA immunoreactivity was well preserved during formalin fixation for 8-24 hr and during fixation delays for 8 hr at room temperature. This indicates that PCNA is stable under conditions routinely encountered in diagnostic surgical pathology and facilitates its potential use as a diagnostic proliferation marker.     

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.     

Visualization of Legionella pneumophila in paraffin sections using hexamine silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 microns. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

Visualization of Legionella Pneumophila in Paraffin Sections using Hexamine Silver

A modification of Gomori's hexamine silver technique is given as a simple, reliable method for the nonspecific demonstration of Legionella pneumophila in paraffin sections. When tested against serogroups I to VI it was found that pretreatment with potassium dichromate rendered L. pneumophila demonstrable by the Gomori-Burtner hexamine silver solution when buffered to pH 7.8. Tissue was fixed in 10% buffered formalin and sections were cut at 3-5 μm. After treatment with 10% potassium dichromate for 1 hour at room temperature, sections are placed in the silver solution at 56 C until they develop a pale golden yellow color, at which point they are checked periodically under the microscope for optimal staining (approximately 3-4 hours). Sections are then toned, fixed and counterstained in 1% neutral red. The L. pneumophila coccobacilli stain black against a clear background, while nuclei stain red/black.     

Rapid Two-Temperature Formalin Fixation

Formalin fixation is a mainstay of modern histopathologic analysis, yet the practice is poorly standardized and a significant potential source of preanalytical errors. Concerns of workflow and turnaround time drive interest in developing shorter fixation protocols, but rapid protocols can lead to poor histomorphology or inadequate downstream assay results. Additionally, assays such as immunohistochemistry for phosphorylated epitopes have historically been challenging in the context of formalin-fixed tissue, indicating that there may be room for improvement in this process that is fundamental to the practice of anatomic pathology. With these issues in mind, we studied basic formalin biochemistry to develop a novel formalin fixation protocol that involves a pre-incubation in subambient temperature formalin prior to a brief exposure to heated formalin. This new protocol is more rapid than standard protocols yet preserves histomorphology and yields tissue that is compatible with an expanded set of downstream clinical and research assays, including immunohistochemistry for phosphorylated epitopes.     

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Abstract

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.     

Microwave fixation of the lung

A technique for microwave fixation of inflated rat lung is described. Conventional intratracheal fixation with instillation of fixative into the airways at a constant pressure results in pressure artifacts as well as flushing and disruption of cells and exudates. Microwave fixation fixes these elements in situ without disruption and thus is valuable when evaluating the distribution of inflammatory infiltrates. Exudative pneumonitis was produced in the rat using intratracheal instillations of either endotoxin or silica and comparisons were made between histologic sections fixed using either standard formalin fixation or microwave fixation.     

Optimizing collagen antigen unmasking in paraffin-embedded tissues

In order to optimize collagen antigen unmasking in paraffin-embedded tissue sections, the effects of various fixatives and duration of fixation in relation to enzyme pretreatment and microwave irradiation for collagen antigen unmasking were studied. A streptavidin--biotin-- peroxidase complex method was used for the immunolocalization of type III and IV collagen antigens. Fixatives and fixation time had significant adverse effects on the immunoreactivity of the antigens. Enzyme pretreatment was found to be superior to microwave irradiation for collagen antigen unmasking. Fixation with paraformaldehyde required shorter enzyme pretreatment and yielded a more enhanced reaction than treatment with formalin and Bouin's fluid. The optimum conditions for type III and IV collagen unmasking were found to be fixation with 4% paraformaldehyde in 0.01 m phosphate-buffered saline, pH 7.4, for up to 3 weeks followed by enzyme pretreatment with 1 mg ml−1 pepsin in 0.01 n hydrochloric acid, pH 2.0, for 30 min (human tissues) or 60 min (rat tissues) at 37°C. It is concluded that collagen antigen unmasking by enzyme pretreatment in tissue sections fixed for a long period of time can be successful if appropriate enzyme(s) and incubation time(s) are employed with regard to the antigen under study and fixative and fixation time used for tissue preparation