Effects of NaOH-PIPES buffer used in aldehyde fixative on alkaline phosphatase activity in rat hepatocytes

Summary Effects of NaOH-PIPES buffer used as a vehicle for aldehyde fixative on alkaline phosphatase (ALPase) activity demonstrated cyto- and biochemically were compared with those of routinely used cacodylate buffer. The reaction products showing ALPase activity demonstrated ultracytochemically were confined to the bile canalicular membranes when cacodylate buffer (0.1 M) was used. However, when PIPES¹ buffer (0.03 M or 0.1 M) was used, the activity was observed on whole membranes of hepatocytes. The activities of the sinusoidal, lateral and bile canalicular membranes were completely suppressed by an addition of 2.5 mM levamisole. Moreover, the same results were obtained when HEPES² or low concentration of cacodylate buffer (0.01 M) was used. Biochemical estimation revealed that much higher activity was retained when PIPES or HEPES buffer was used as compared with that when cacodylate buffer was used. Maximum preservation of ALPase activity was obtained when PIPES buffer was used. Cacodylate buffer showed an inhibitory effect on the hepatic ALPase activity in proportion to the buffer concentration.In conclusion, PIPES buffer preserves the alkaline phosphatase activity much better and is a better vehicle for the aldehyde fixatives in alkaline phosphatase cytochemistry.1 PIPES piperazine-N,N-bis (2-ethanesulfonic acid) - 2 HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid This study was supported by a Grant-in Aid for Encouragement of Young Scientists from the Ministry of Education, Science and Culture, the Japanese Government (No. 57770012)     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation–Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds

Summary Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study, I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32–34 s, final temperature between 40° C and 47° C.     

Impact of glutaraldehyde versus glutaraldehyde-formaldehyde fixative on cell organization in fish corneal epithelium

The purpose of this study was to examine the impact of a low osmolality glutaraldehyde fixative and a high osmolality glutaraldehyde-formaldehyde fixative on the structural organization of a tissue that could be exposed to low and high osmolality environments. The corneas of freshwater trout were prepared for transmission and scanning electron microscopy using either a fixative of 2% glutaraldehyde in 60 mM cacodylate buffer (pH 7.8, 260 mOsm/l) or a fixative prepared by adding 2.5% glutaraldehyde to a solution of 1% formaldehyde and buffering the solution with 0.1 M cacodylate (pH 7.6, 850 mOsm/l; Karnovsky-type fixative). The corneal epithelial cell layer thickness was greater after glutaraldehyde compared to glutaraldehyde-formaldehyde fixation (67 vs 55 mum), as was the thickness of the superficial cells (5.1 vs 3.4 mum) and basal cells (43 vs 38 mum). The intermediate (wing) cells of the epithelium were, however, less thick after glutaraldehyde fixation (15 vs 18 mum). The width of the squamous, intermediate and basal cells was greater following glutaraldehyde fixation with the effect being greatest in the superficial layers and insignificant at the level of the basal cells. The results show that chemical fixatives with extremes of osmolality cannot only produce different cell sizes in a tissue but also determine the overall organization of the cells in a positional-dependent fashion.     

Fixation of vervet monkey oral mucosa for ultrastructural investigation--TEM

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecus pygerythrus) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolarities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

Double immunohistochemical labeling technique applied to different types of cytokeratins in epithelial proliferations of the breast

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.     

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein-Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was "gentle" fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as "gentle" cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or--if one does not have unfixed tissues--solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.     

Evaluation of fixative solutions for ultrastructural analysis of brown spider Loxosceles intermedia (Araneae: Sicariidae) tissues.

In view of the widely varying compositions of fixative solutions used for studying spiders, five different fixative formulas were tested for fixing male brown-spider (Loxosceles intermedia) gonad tissues. The brown spider represents a public health problem in Curitiba (Paraná State, Brazil). Morphological study of its gonads may aid in understanding the reproductive strategies of this species, and possibly in developing a reproduction control program. The fixatives tested contained glutaraldehyde alone or combined with paraformaldehyde, and the buffers cacodylate or phosphate, with or without the addition of sucrose or sodium chloride as osmolytes. Those containing 2.5% glutaraldehyde and 2% paraformaldehyde in 100 mM phosphate buffer with 200 mM sucrose, or in 200 mM sodium cacodylate, satisfactorily preserved mitochondria, the Golgi apparatus, and the membranes in general. These formulas were nearly isosmotic (439 mOsm/kg H2O and 455 mOsm/kg H2O respectively) to brown spider hemolymph (478 mOsm/kg H2O). With respective to the fixative agents, a glutaraldehyde-paraformaldehyde combination resulted in optimal fixation of Loxosceles intermedia cells. For other species of spiders, hemolymph osmolality should be considered, but the fixative formulas cited above would also probably yield good results.     

Fixation of Vervet Monkey Oral Mucosa for Ultrastructural Investigation-Tem

This study was undertaken to determine optimal fixation procedure for vervet monkey (Cercopithecui pygerythrta) oral mucosa. Perfusion and immersion fixation were investigated using glutaraldehyde and glutaraldehyde-paraformaldehyde fixatives with either a phosphate or sodium cacodylate buffer as vehicle and with osmolalities varying from 2010 to 320 mosm. Good fixation could not be obtained uniformly or consistently by perfusion. Vervet monkey oral mucosa is best fixed by first perfusing the head and neck of the animal with 250-500 ml 0.9% saline containing Procaine-HCl and heparin, followed by decapitation and immersion of the head in a 2.5% glutaraldehyde: 2% paraformaldehyde: 0.02 M sodium cacodylate buffered fixative (900 mosm) at 4 C for 24 hr.     

EFFECTS OF FIXATIVES ON THE ULTRASTRUCTURE OF PHYSODES IN VEGETATIVE CELLS OF SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE,PHAEOPHYTA)1

The effects of different glutaraldehyde-osmium fixation schedules on the ultrastructure of the vegetative cells from the meristematic regions of Scytosiphon lomentaria (Lyngbye) Link fronds are described. The best overall preservation of cell structure was obtained with a 2 h fixation in 2.5–3.5% glutaraldehyde in 0.1 M cacodylate buffered seawater (pH 7.0), followed after washing by 1 h post fixation in 1% osmium tetroxide. The addition of 1% caffeine to the glutaraldehyde fixative resulted in better retention and spatial localization of the electron dense phenolic deposits within the cells. Particular attention was paid to the effects of the various fixation schedules on the electron-dense material within the cells and the images obtained were compared with previous accounts of brown algal cells. It is proposed that the term physode should be restricted to the discrete electron dense spherical bodies within the vacuoles and not applied to electron dense material in general. Although the organization of Scytosiphon cells was similar to that previously reported in the Scytosiphonaceae, the organization of the plasmodesmata into pit fields is at variance with previous accounts.     

Microwave-stimulated glutaraldehyde and osmium tetroxide fixation of plant tissue: ultrastructural preservation in seconds.

Microwave-enhanced fixation of animal tissues for electron microscopy has gained in interest in recent years. Attempts to use microwave irradiation for the preparation of plant tissues are rare. In this study; I report on microwave conditions which allow a high quality preservation of plant cell structure. Tissues used were: internodes of Chara vulgaris, leaves of Hordeum vulgare, root tips of Lepidium sativum. Microwave irradiation was done with a commercial microwave oven (Sharp R-5975). Fixatives used were: 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2 and 1% osmium tetroxide in veronal/acetate buffer, pH 7.2. Conventional fixations with glutaraldehyde/osmium were compared with microwave fixations. Examinations of thin sections showed that microwave fixation (glutaraldehyde or sequential aldehyde/osmium) is an attractive and rapid alternative method for processing plant tissues for electron microscopy. The optimal conditions found were: microwave oven at power level 50 W, 6.5 ml of fixative solution, irradiation times between 32-34 s, final temperature between 40 degrees C and 47 degrees C.     

Enhanced preservation of endosecretory granules in PP-cells of the canine pancreas

Standard fixation techniques commonly used for light and electron microscopic studies have resulted in reported differences in the ultrastructural appearance of endosecretory granules of the pancreatic polypeptide (PP) cell. To clarify these differences, canine pancreatic tissues of intact and cultured pseudoislets were studied using a variety of ingredients, additives and fixatives in an effort to better preserve the endosecretory granules of PP cells. Results show that preservation of PP granules is enhanced by addition in zinc chloride (0.5%) to a glutaraldehyde-paraformaldehyde fixative in 0.1 M cacodylate buffer, followed by osmium tetroxide fixation. This fixative is recommended for all light and electron microscopic studies of the pancreatic polypeptide cell.     

Morphometric analyses of the electric organ of Torpedo: The influence of different fixative modes on the vesicle diameter

Summary The influences of various fixatives on the vesicle size of the electric organ of Torpedo marmorate were investigated. Thin section and freeze etched preparations were examined under the electron microscope.In thin section increased vesicle diameters were observed compared with the freeze etched preparations. The same experiments in different torpedo fish led to significantly different vesicle sizes observed. Variations of the molarity, the pH and osmolarities result in particularly high differences in vesicle diameters.Using Karnovsky's method (1965) and a fixative consisting of 2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.2, results in vesicle sizes comparable to those reported by other authors.Results obtained from freeze etched preparations are not comparable in general with results from thin section experiments with the same fixative.     

Biochemical aspects of aldehyde fixation and a new formaldehyde fixative.

In this paper, it is assumed that tissue fixation is a process in which the proteins become less soluble and catabolic reactions stop. With this definition in mind, 2.5 and 5% glutaraldehhde and 4% formaldehyde in 0.1 M potassium phosphate buffer, pH 7.4, were compared with a new fixative, bicarbonate-formaldehyde. The following results were obtained. (I) With 2.5 and 5% glutaraldehyde, the solubility of tissue proteins were not decreased unifromly, and tissue glycogen was poorly preserved. (2) 4% formaldehyde in potassium phosphate buffer gave relatively good results. (3) Bicarbonate-formaldehyde decreased the solubility of tissue proteins reliably and preserved tissue glycogen perfectly. Histologically, it yielded excellent results. Since glutaraldehyde alters the properties of proteins substantially (Hopwood, 1972; Habeeb & Hiramoto, 1968), and since the natural appearance of tissues depends on native tissue proteins, formaldehyde-containing fixatives, in particular bicarbonate-formaldehyde, are preferable to glutaraldehyde-containing fixatives for all tissue preparative techniques. However, it is important that the fixation time in formaldehyde is kept short.     

Commercial Formalin Substitutes for Histopathology

We compared the performance of six commercial fixatives proposed to be formalin substitutes with the performance of buffered formalin, Clarke's ethanol-acetic acid, and ethanol, using rat liver, small intestine, and kidney. We investigated the rate of penetration, mode of fixation, extent of protein and structural immobilization, quality of histology and cellular structure following routine dehydration and paraffin embedding, and performance as a fixative for immunohistochemistry. Furthermore, we evaluated the effects of the various fixatives on ultrastructure. Only buffered formalin performed equally well on all tissues tested. While several of the commercial fixatives appeared to preserve liver tissue at 200, the preservation of kidney, intestinal villi, and smooth muscle was unacceptable. Histological distortion, cell shrinkage and vacuolization were prominent when the substitutes or ethanol were used. In contrast, these artifacts were found occasionally and to a minor degree when buffered formalin or Clarke's fixative were used. Immunohistochemistry demonstrated a total loss of low molecular weight antigen (serotonin) and patchy reactions for high molecular weight antigens for all fixatives except buffered formalin. The best immunostaining was obtained by combining formalin fixation with antigen retrieval. We conclude that none of the proposed commercial substitutes for buffered formalin are adequate for critical histology or histopathology.     

Effects of Fixation and Tissue Processing on Immunohistochemical Demonstration of Specific Antigens

Identification of biomarkers in archival tissues using immunochemistry is becoming increasingly important for determining the diagnosis and prognosis of tumors, for characterizing preinvasive neoplastic changes in glandular tissues such as prostate, for evaluating the response of tumors and preinvasive neoplastic changes to certain therapies (i.e., as a surrogate intermediate end point), for selecting patients who are candidates for specific therapies (e.g., immunotherapy) and for retrospective studies. For detecting specific biomarkers it is important to understand the limitations imposed by the fixation methods and processing of the tissues. This study was designed to determine the effects of fixation on the detection in archival paraffin blocks of selected antigens postulated to be important in tumor biology. We evaluated the antigens TGFα, p185^erbB-2, broad spectrum keratins, p53, and TAG-72 (B72.3). Fixatives evaluated included standard preparations of neutral buffered formalin, acid formalin, zinc formalin, alcoholic formalin, ethanol, methanol, and Bouin's fixative. We found that in general neutral buffered formalin is the poorest fixative for maintaining antigen recognition by immunohistochemistry and that no single fixative was best for all antigens. The dehydrating (coagulant) fixatives (e.g., ethanol and methanol) preserved immunorecognition of p53 and broad spectrum keratins best while the slow cross-linking fixatives (e.g., unbuffered zinc formalin) were best for demonstrating TGFα and p185^erbB-2. Fixatives other than neutral buffered formalin produced equivalent recognition of the epitope of TAG-72 by B72.3. In formalin fixed archival tissues, only a portion of the antigen signal can be detected by routine immunohistologic methods.     

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.     

Cytochemical demonstration of adenylate cyclase in cardiac muscle: effect of dimethyl sulfoxide.

Adenylate cyclase (AC) activity was evaluated after perfusion fixation of rat and dog myocardium with 4% paraformaldehyde (PFA), 2% glutaraldehyde (GA) or a combination of both, in cacodylate buffer. Dimethyl sulfoxide (DMSO) was added to the fixatives and its effect on the preservation of cell organelles and enzyme activity was determined. Adenylate cyclase activity was preserved best after fixation with 4% paraformaldehyde but this fixative did not provide for optimal maintenance of structure. Prefixation with 2% glutaraldehyde and 5% dimethyl sulfoxide provided the most effective preservation of both structural and enzymatic integrity. Precipitation of lead diphosphoimide was the morphologic indicator of sites of adenylate cyclase activity. The most intense precipitate was in the lumen of junctional sarcoplasmic reticulum in close contact with T-tubules and in subsarcolemmal cisternae. Evidence of activity was also seen on the intracellular aspect of the sarcolemmal membrane and in the nexus segment of the intercalated discs. Alloxan was effective as an inhibitor of adenylate cyclase activity only if the concentration of the activating substance sodium fluoride (NaF) was 20 mM or lower.     

大鼠睾丸组织固定法的优选研究

目的选择一种最优势、合理的固定方式以提高对睾丸组织制片效果,以配合不同种类的科学研究。方法选用10％甲醛溶液、NBF—Bouin’s、Bouin’s和改良Davidson’s四种不同的固定液对大鼠睾丸进行充分固定后,制作石蜡切片,进行HE染色,比较不同固定液中的睾丸组织学形态的差异;利用糖原特殊染色（PAS）,探讨不同固定液对睾丸糖原观察的影响;采用免疫组织化染色,测评睾丸组织内雄激素受体的固定效果。结果改良Davidson’8固定液较NBF—Bouin’s引起的曲细精管萎缩轻,形态更为清晰,用免疫组织化学方法检测雄激素更为敏感,并且改良Davidson＇s固定液在需要对精子发生进行分期时,其PAS染色的效果与Bouin＇s液固定后等同。结论与苴守圈常浦相№曲冉David0Rnn浦对女宙奥由的圈索特罩掂杯     

Evaluation of zinc salt based fixatives for preserving antigenic determinants for immunohistochemical demonstration of murine immune system cell markers

Conventional aldehyde based fixatives produce good morphological preservation. However, owing to their cross-linking mechanism of action, epitope loss may occur during fixation compromising the tissue for subsequent immunohistochemical (IHC) analysis. IHC is an important tool for characterizing antigen, cytokine and cytomorphological markers. The increasing use of mouse models for study of pathogenesis has highlighted the need to investigate alternative fixatives. In the study reported here, tissue samples from RIII mice with immune mediated lesions, Mycobacterium bovis infected mice, and uninfected control mice were fixed in either zinc salt fixative or buffered formalin, then tested for IHC using a panel of antibodies (CD3, CD4, CD8, CD45, CD54, F4/80, Interferon-gamma and MIP2). Zinc salt fixation preserved processing-sensitive murine cell markers (CD4, CD8 and CD54) and improved the intensity of immunolabeling for CD45, F4/80 and CD3. Buffered formalin failed to preserve any of the processing-sensitive murine epitopes for demonstration by subsequent IHC.