Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears.

Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic integrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2--10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.     

Effect of fixation on the antigenicity of human lactoferrin in paraffin-embedded tissues and cytocentrifuged cell smears

Summary Immunoperoxidase techniques were used to study the preservation of the antigenicity of human lactoferrin (LF) of polymorphonuclear neutrophils (PMN) and various exocrine glandular cells in paraffin-embedded tissue blocks and cytocentrifuged cell smears. Tissues fixed in Carnoy's fluid in contrast to other fixatives used, showed good preservation of LF antigenicity irrespective of the fixation time. Cell smears fixed in Carnoy's fluid showed diffuse nuclear and cytoplasmic staining for LF, although morphologic intergrity was poorly preserved. Granular cytoplasmic staining for LF with no staining of nuclei was seen in cell smears fixed in buffered formol acetone for 2–10 min. The nature of nuclear LF staining and future applications of the present methods are discussed.     

间接免疫过氧化物酶技术鉴定猪和牛的肥大细胞

用小鼠抗人肥大细胞类胰蛋白酶单克隆抗体ＡＡ１,ＡＡ３及ＡＡ５的间接免疫过氧化物酶技术对经Ｃａｒｎｏｙ液或中性缓冲福尔马林固定的猪和犊牛空肠,舌及胸腺的石蜡切片进行了免疫染色。对猪和牛的肥大细胞特异性免疫染色与常规的组织化学染色的结果进行了比较。     

Microwave stabilization versus chemical fixation. A morphometric study in glycolmethacrylate- and paraffin-embedded tissue

Summary A morphological and morphometrical study was performed on testicular cells after microwave stabilization of the tissue while immersed in phosphate buffered saline (PBS), 0.9 NaCl or Tris-HCl. Fixation in Carnoy's fluid without irradiation was chosen as a control chemical fixation method. After microwave stabilization or chemical fixation, the testes were embedded in paraffin or in plastic (glycolmethacrylate).An excellent morphology, comparable to that after chemical fixation in Carnoy's fluid, was observed in the plastic sections of tissue irradiated in PBS or NaCl, even when the sections were subsequently treated with an aggressive reagent at high temperature, required for the Feulgen reaction. The nuclear area of the microwave-stabilized Sertoli cells was 37–46% smaller in haematoxylin-eosin stained, paraffin sections in comparison with that in the glycolmethacrylate sections. The microwave-stabilized, paraffin-embedded tissue was much more vulnerable to the hot HCl treatment of the Feulgen staining than the chemically fixed tissue, resulting in an additional 10–20% decrease in nuclear size. The latter finding is particularly important for quantitative microscopy, where the Feulgen staining method is often employed.     

A comparative study of quantitative stains for DNA in image cytometry.

In this study we examined the reproducibility of several stains used to measure nuclear DNA by image cytometry. The specimens were touch preparations of liver and testis from mouse and liver, intestine and brain from rat, fixed in either neutral formalin or Carnoy's solution. The tested stains included four Feulgen methods (pararosaniline, azure-A, thionin and acriflavine), the gallocyanine-chromalum stain and two fluorescent stains (acridine orange and propidium iodide). Absorbance measurements employed a video image analysis system; fluorescence measurements were from a scanning microspectrophotometer. The acriflavine-Feulgen stain was analyzed for both absorbance and fluorescence. All seven stains were quantitative for DNA and gave reproducible results. The absorbance measurements had a lower coefficient of variation (CV) than the fluorescence values. In a nested analysis of variance of the pararosaniline Feulgen stains, cell-to-cell variability accounted for 67% of the total variance; slide-to-slide, 9%; and batch-to-batch, 24%. These values did not change significantly when the staining was performed in an automatic staining machine. For DNA analysis using image cytometry, we conclude that the Feulgen staining technique is the most useful. In particular, acriflavine-Feulgen-stained cells fixed in Carnoy's fluid give the least variation between measurement values and the most accurate ratios between the separate ploidy groups. For fluorescence cytometry we recommend Carnoy's fixation and the acriflavine-Feulgen stain because of its narrow CV as compared to acridine orange and propidium iodide.     

The importance of tissue fixation for light microscopic immunohistochemical localization of peroxisomal proteins: the superiority of Carnoy's fixative over Baker's formalin and Bouin's solution

We have compared the effects of fixation with three commonly used fixatives upon preservation of the antigenicity of six peroxisomal proteins in rat liver using both immunohistochemical staining and Western blotting of fixed tissue extracts. The immunoreactivity of all six peroxisomal proteins was well preserved and peroxisomes were clearly identified in material fixed in Carnoy's fixative. Moreover, the corresponding proteins stained well in Western blots prepared from extracts of Carnoyfixed material. The intensity of the immunohistochemical staining was reduced at different rates for individual peroxisomal proteins after fixation in Baker's formalin, but peroxisomes were still well visualized with antibodies to catalase and some -oxidation enzymes. No evidence of immunohistochemical staining for any peroxisomal antigens was obtained after fixation in Bouin's fluid. For detection of the antibody binding sites in Carnoy's fixed material, the avidin-biotin-peroxidase complex (ABC) with aminoethyl carbazole as chromogen was found to be superior to the methods of peroxidase-antiperoxidase/diaminobenzidine and protein A-gold with silver intensification. Using Carnoy-fixative and the ABC-method, we demonstrate light microscopic immunohistochemical localization of peroxisomal antigens in several rat tissues as well as in human post-mortem liver.     

A Modified Technic for Salivary-Gland Chromosomes

This technic was worked out for making permanent mounts of salivary-gland squashes of strains and species of Drosophilidae, which yield material difficult to work with. It includes a prior fixation in a modified Carnoy's fluid, staining in solutions of orcein in 60% or 70% acetic acid, part dehydration in the vapor and liquid of a 95% solution of alcohol, and direct mounting in Euparal by means of the drainage-method.     

Whole Mounts of Small Embryos Attached Directly to Glass Slides

Suspensions of sea urchin embryos spread over the surface of glass slides were attached to the glass by a rapid coagulation of the surface with alcohol. This was done either by dipping the glass slides into absolute alcohol or by a short exposure to alcohol vapor. Thereafter the slides were immediately transferred to the fixative. A suitable procedure includes fixation with Carnoy's fluid (alcohol, chloroform, acetic acid; 6:3:1) and staining with Gomori's hematoxylin after acid hydrolysis.     

A combination staining method for fibers and cell bodies of the urodele central nervous system

A simple method for staining nerve cells and fibers of the salamander central nervous system is described. The procedure employs Carnoy's fixation followed by Protargol inpregnation and Nissl staining. This technique permits the simultaneous observation of intracellular neurofibrils, neuronal processes and basophilic components of the neuron. In addition, it eliminates the need to stain alternate sections with separate procedures to view the various components of the urodele central nervous system.     

A Combination Staining Method for Fibers and Cell Bodies of the Urodele Central Nervous System

A simple method for staining nerve cells and fibers of the salamander central nervous system is described. The procedure employs Carnoy's fixation followed by Protargol impregnation and Nissl staining. This technique permits the simultaneous observation of intracellular neurofibrils, neuronal processes and basophilic components of the neuron. In addition, it eliminates the need to stain alternate sections with separate procedures to view the various components of the urodele central nervous system.     

The use of OsO4 as fixative for Feulgen-stained preparations

OsO(4) has many advantages over Carnoy's fixative mixture for the Feulgen nuclear staining in the protozoan Tokophrya infusionum. While Carnoy's fluid used prior to the Feulgen reaction produces shrinkage of the macronucleus and coarse clumping of its chromatin bodies, OsO(4) preserves faithfully the size and shape of the macronucleus and its chromatin material. This finding seems to be of special importance in view of the fact that electron microscopy relies on OsO(4) fixation. The satisfactory preservation of structured detail in Feulgen-stained preparations is of importance for the correlation of histochemical and morphological information.     

Microwave irradiation improvements in the silver staining of the nucleolar organizer (Ag-NOR) technique

The well-know technique of silver staining of the nucleolar organizer (Ag-NOR) is improved in contrast, selectivity and speed when performed with microwave irradiation. The Ag-NOR technique is a very useful tool for studies on the functional morphology and molecular architecture of the nucleolus, and is reputed to be one of the best techniques for diagnosis and prognosis of cancer lesions. To test the generality of the enhancing effects. our study has involved the use of both mammalian and plant cells. Two steps in the process are improved quantitatively by microwave irradiation: fixation and staining itself. Fixation with the ethanol-based reagent, Kryofix, for 3 min in the microwave oven, resulted in good structural preservation at the optical level, and enhanced the contrast and selectivity of silver staining. On the contrary, we found that neither glutaraldehyde fixation, nor a treatment of sections with Carnoy's solution, improved Ag-NOR staining. After an analysis of the effects of the different substances involved in sample preparation, we conclude that ethanol is an essential factor for fixation for nucleolar staining, particularly if aldehydes are eliminated from fixative solutions. The process of staining was performed with a drop of staining solution on a semithin section of plastic-embedded tissue intthe microwave oven for 1 min. Staining under these conditions always improved the visualization of nucleoli, regardless of the fixation procedure. Therefore, microwave irradiation at both steps is recommended for giving the best results. Microwave irradiation probably enhances fixation by controlled heat, whereas the increase in reactivity of the staining solution is a direct effect by the microwaves on the silver ions themselves. We used this method to study nucleolar materials during mitosis in proliferating plant cells. Current applications of Ag-NOR staining can be improved with this technical modification.     

Effects of different fixatives on beta-galactosidase activity.

beta-Galactosidase (beta-Gal) staining is widely used to demonstrate specific gene expression during evaluation of gene targets in vivo. This technique is extremely sensitive to fixation. Optimal fixation conditions are necessary to obtain the maximal beta-Gal activity. In this experiment, Carnoy's and three different aldehyde fixatives were used at different temperatures and over different time points. Kidneys from LacZ-stop-human alkaline phosphatase (ZA/P) double reporter mice were used to generate positive material for the experiment. The results show that glutaraldehyde combinative solution (LacZ) produced the most consistent and reliable results. Paraformaldehyde and formaldehyde were effective as fixatives only at 4C for a period of less than 4 hr, and Carnoy's solution destroyed beta-Gal activity.     

The Use of OsO4 Fixative for Feulgen-Stained Preparations

OsO₄ has many advantages over Carnoy's fixative mixture for the Feulgen nuclear staining in the protozoan Tokophrya infusionum. While Carnoy's fluid used prior to the Feulgen reaction produces shrinkage of the macronucleus and coarse clumping of its chromatin bodies, OsO₄ preserves faithfully the size and shape of the macronucleus and its chromatin material. This finding seems to be of special importance in view of the fact that electron microscopy relies on OsO₄ fixation. The satisfactory preservation of structured detail in Feulgen-stained preparations is of importance for the correlation of histochemical and morphological information.     

Two Organic Fixatives for Acid Mucopolysaccharides

Cetyl pyridinium chloride, 0.5% in 4% aqueous formaldehyde and 5-aminoacridine hydrochloride, 0.4% in 50% aqueous ethanol, have been tested as fixatives for acid mucopolysaccharides in a variety of tissues. These solutions are superior to 4% aqueous formaldehyde, Carnoy's fluid and basic lead acetate for this purpose and also give good nuclear fixation. Tissues containing these mucopolysaccharides are particularly well defined with the acridine-ethanol fixative.     

Processing techniques for the demonstration of myelin basic protein in paraffin-embedded optic nerve: an immunoperoxidase study of the developing albino rat

A comparison was made of the effects of various fixation and processing conditions upon the antigenicity of myelin basic protein (MBP) in sections of paraffin-embedded optic nerve from the developing albino rat as judged by the unlabeled peroxidase-antiperoxidase technique. The fixatives used were: Perfix, 4% and 2% buffered paraformaldehyde (pH 7.4), 10% buffered formalin (pH 7.4); Bouin's, Clark's, and Carnoy's fixatives, and 20% formalin in a solution of HgCl2 that had been saturated at 1 degrees C. Perfix appeared to be the best fixative for the preservation of morphology and MBP antigenicity during the early stages of myelinogenesis but was not satisfactory during the later stages. The buffered aldehydes were slightly more destructive of MBP antigenicity than was Perfix, but they produced satisfactory results following the first postnatal week. Bouin's fixative was similar in effect to the buffered aldehydes, but nonspecific background staining was higher. HgCl2/formalin, Clark's and Carnoy's fixatives were unsuitable. No differences were noted in staining between material processed for embedding using 5, 30, or 60 min schedules.     

On the influence of fixations on the normal hydration of rabbit cornea and the total amount of acid mucopolysaccharides in the stroma

Summary The authors studied the influence of fixations on the normal hydration of the rabbit cornea and the total amount of acid mucopolysaccharides (AMPS) in the stroma. The following fixatives were used: formol-calcium chloride at 19° C for 24 hours, formolcetylpyridinium chloride (CPC) at 19 and 28° C for 48 hours, Lillie's fixation at 19° C for 24 hours and Carnoy's fluid at 19° C for 30 minutes. The sections of the corneae were stained with Alcian blue, colloidal Fe³⁺ in the modification according to Rinehart and Abu'l Haj and with toluidine and methylene blue. The amount of AMPS was determined with the method of Rondle and Morgan and the total hydration of the stroma by weighing the corneae before and after using different fixative fluids and by calculation of obtained values on dry weight.The best results were obtained by using formol-CPC at 28° C. At the ordinary room temperature (±19° C) it was the poorest fixation, however, as the corneae in this solution became hydrated. Formol-calcium chloride was the second in the row and it was much better than Lillie's and Carnoy's fluid.The amount of AMPS in the stroma was not essentially changed by the effect of fixatives. Within 24–48 hours formol-CPC at 28° C retained the normal content and formol-calcium chloride caused the 11% decrease of AMPS maximally. The loss of AMPS after other fixatives was minimal.The intensity of staining with cationic dyes in paraffin sections was different after individual fixatives and after the kind of their application and was dependent chiefly on the state of hydration of the corneal stroma: It is impossible to interpret the results of staining reactions in terms of the quantity of AMPS as it was hitherto done.     

不同固定液和染色方法显示山羊子宫内肥大细胞效果的比较

目的探讨用不同固定液和染色方法对显示处于间情期山羊子宫肥大细胞的影响。方法用四种不同的固定方法,应用改良甲苯胺蓝（MTB）染色法和阿尔辛蓝-番红花红（AB-S）染色法显示处于间情期山羊子宫肥大细胞。结果山羊子宫组织采用Carnoy氏液固定,MTB和AB-S染色对所有的肥大细胞均可获得良好的染色反应,但10％中性福尔马林,4％多聚甲醛,Bouin氏液固定的组织仅有少量肥大细胞着染。结论MTB和AB-S染色法均是山羊子宫肥大细胞良好的染色方法。     

鸡中枢淋巴器官肥大细胞的组织化学与形态学

对哺乳动物的,特别是啮齿动物和人类肥大细胞已有了比较深入的研究, 但关于家禽肥大细胞的研究很少.本研究旨在阐明鸡中枢淋巴器官中肥大细胞的组织化学与形态学特征.本研究证实Carnoy 氏液是鸡肥大细胞的优良的固定液,而中性缓冲福尔马林(NBF) 却阻断了大多数肥大细胞的着染力.甲苯胺蓝和阿尔新蓝是鸡肥大细胞的良好的染料,但阿尔新蓝能使更多的肥大细胞着染,虽然其也可使杯状细胞着染.作者的一种新的染色法, 长时间阿尔新蓝染色(LAB-S)可用于NBF固定的组织中肥大细胞的染色,因为其着染的细胞数与Carnoy 氏液固定甲苯胺蓝染色的细胞数无显著差异(P<0.001).在胸腺髓质中见有大量的肥大细胞,而胸腺皮质仅可见个别肥大细胞位于血管周围及小叶间结缔组织中.腔上囊的皮质与髓质中很少见有肥大细胞.肥大细胞有血管周围分布的倾向,但一个有趣的发现是血管内偶尔也有个别肥大细胞.电镜下可见肥大细胞的胞浆颗粒内充满无定形的颗粒状基质,但其电子密度有的较高,有的较低.少数胞浆颗粒内有旋涡状及网状亚微结构.但未见有人类肥大细胞胞浆颗粒内特征性的晶格状和卷轴状的亚微结构,也未见到在绵羊肥大细胞中描述过的特殊亚微结构.     

Squash Method for Meiosis in Ovules

The ordinary Feulgen or acetic-lacmoid squash tech-nic following fixation in freshly made Carnoy's fluid (alcohol, 6: chloroform, 3: glacial acetic acid, 1), provides an easy and reliable method of studying meiosis in ovules. After fixation for 1 day, the material was hardened in 95% ethyl alcohol for 1-2 days and taken to water by gradual hydration. For staining by the Feulgen method, the material was hydrolyzed 8-10 minutes in 1 N HO at 58-60°C., followed by staining in decolorized leuco basic fuchsin for 2 hours. The staining was intensified by transferring the material to water. After 15-20 minutes the water was replaced by 45% acetic acid. For staining by acetic-lacmoid, the ovules after fixation, hardening and hydration were transferred to standard acetic-lacmoid stain to which was added 1 drop of 1 N HCl to every 10 drops of stain. Gentle heat was applied till the stain started to give fumes. After allowing 20 minutes at room temperature the material was transferred to fresh acetic-lacmoid. Some 6-12 ovules were mounted either in a drop of 45% acetic acid or acetic-lacmoid, depending upon the Feulgen or acetic-lacmoid staining respectively. Gentle and repeated tapping over the cover glass by a blunt needle loosened the cells of integument and nucellus and finally left the megaspore mother cells undergoing meiosis, fully exposed to view. The process was carried out under constant observation using the low power of the microscope. The desired amount of flattening was brought about by light pressure over the cover glass and gentle heating. The preparations were made permanent by dehydrating in ethyl alcohol and mounting in Euparal.