Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy

Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible. In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast.     

Improved detection and quantification of the (immuno) peroxidase product using reflection contrast microscopy

Summary Reflection contrast microscopy (RCM) is a sensitive tool to detect minor amounts of precipitated diaminobenzidine (DABox) in immunoperoxidase stained specimens. One of the main issues in immunocytochemistry is the ongoing need for more sensitive and quantitative techniques. Therefore we applied RCM, using a new simple model system, to methods previously described for increased sensitivity in immunocytochemistry with bright field microscopy. Addition of imidazole was found the most sensitive method and addition of Nickel and Cobalt ions gave the most enhanced colour intensity. Variation of the enzyme reaction parameters yielded a continuous increase in reflection with time. This was then discussed in view of other model studies of peroxidase kinetics. A quantitative relationship between the amount of peroxidase and the reflection of DABox was observed, indicating that quantitative immunoperoxidase studies with RCM are feasible.In situ hybridization (ISH) was then used as a useful biological model for RCM to test the optimal conditions for DAB staining found in the model system (high concentrations of DAB and peroxidase and 2 h incubation time). There was no background staining in the model system, also after prolonged incubation time. The ISH experiments showed that the contrast (ratio) between specific signal and chromosome background did not increase in time, whereas only the use of high avPO concentrations yielded the highest contrast.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy

Summary One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

New sensitive light microscopical detection of colloidal gold on ultrathin sections by reflection contrast microscopy. Combination of reflection contrast and electron microscopy in post-embedding immunogold histochemistry.

One simple post-embedding method for combined light- and electron microscopy is presented. Different types of antigens in normal rat and mouse kidneys as well as in tissues from cases of experimental induced nephritis were stained after Lowicryl K4M embedding by an immunogold (silver) method. The (silver-enhanced) gold particles were visualized by light microscopy, e.g. bright-field (BFM)- and reflection contrast (RCM) microscopy, as well as by electron microscopy. The potentials of RCM visualization in this field were investigated, resulting in the successful detection of colloidal gold (15 nm) particles, or silver enhanced gold particles, on ultrathin sections. Furthermore, an increased detection sensitivity of RCM compared with BFM together with an increase in the sensitivity of the immunostaining by RCM visualization was found. The different ways to use RCM, alone or in combination with bright-field- or phase contrast microscopy for visualization of plastic sections varying in thickness, type of plastic and staining, are discussed.     

Reflection contrast microscopy

Summary Reflection contrast microscopy (RCM) has proven to be a useful tool for the study of living cells (Ploem 1975). Due to the effective suppression of aspecific reflected light by polarization optics combined with a quarter lambda plate at the front lens of the objective, low intensity reflection signals originating from minor amounts of precipitated diaminobenzidine (DABox) in immunocytochemically stained specimens, can be made visible. RCM has been successfully applied in demonstrating single copy nucleic acid sequences using in situ hybridization procedures (Landegent et al. 1984).We have systematically studied the aspects of image formation of DABox by RCM by using a model system consisting of glass slides coated with peroxidase containing protein layers to determine the conditions for optimal sensitivity of this detection method. Moreover, investigations were performed to study the relationship between the amount of reflected light and DABox depending on the thickness of the object. Both theoretical and practical evidence is obtained to show that DABox detection by RCM is based on interference phenomena occurring in the layer of DABox, and less on selective reflection. This restricts the type of specimen which can be used for sensitive detection of DABox by RCM. Consequently, in ultrathin (40 nm) sections osmificated DABox was visualized in peroxidatic positive cell organelles with high contrast and resolution. Similar results were obtained with immunoperoxidase stained material embedded in Lowicryl under conditions that did not allow visualization of the staining product by bright field microscopy.     

Reflection contrast microscopy. Visualization of (peroxidase-generated) diaminobenzidine polymer products and its underlying optical phenomena

Reflection contrast microscopy (RCM) has proven to be a useful tool for the study of living cells (Ploem 1975). Due to the effective suppression of aspecific reflected light by polarization optics combined with a quarter lambda plate at the front lens of the objective, low intensity reflection signals originating from minor amounts of precipitated diaminobenzidine (DABox) in immunocytochemically stained specimens, can be made visible. RCM has been successfully applied in demonstrating single copy nucleic acid sequences using in situ hybridization procedures (Landegent et al. 1984). We have systematically studied the aspects of image formation of DABox by RCM by using a model system consisting of glass slides coated with peroxidase containing protein layers to determine the conditions for optimal sensitivity of this detection method. Moreover, investigations were performed to study the relationship between the amount of reflected light and DABox depending on the thickness of the object. Both theoretical and practical evidence is obtained to show that DABox detection by RCM is based on interference phenomena occurring in the layer of DABox, and less on selective reflection. This restricts the type of specimen which can be used for sensitive detection of DABox by RCM. Consequently, in ultrathin (40 nm) sections osmificated DABox was visualized in peroxidatic positive cell organelles with high contrast and resolution. Similar results were obtained with immunoperoxidase stained material embedded in Lowicryl under conditions that did not allow visualization of the staining product by bright field microscopy.     

Quantification and visualization of coordination during non-cyclic upper extremity motion

There are many design challenges in creating at-home tele-monitoring systems that enable quantification and visualization of complex biomechanical behavior. One such challenge is robustly quantifying joint coordination in a way that is intuitive and supports clinical decision-making. This work defines a new measure of coordination called the relative coordination metric (RCM) and its accompanying normalization schemes. RCM enables quantification of coordination during non-constrained discrete motions. Here RCM is applied to a grasping task. Fifteen healthy participants performed a reach, grasp, transport, and release task with a cup and a pen. The measured joint angles were then time-normalized and the RCM time-series were calculated between the shoulder-elbow, shoulder-wrist, and elbow-wrist. RCM was normalized using four differing criteria: the selected joint degree of freedom, angular velocity, angular magnitude, and range of motion. Percent time spent in specified RCM ranges was used as a composite metric and was evaluated for each trial. RCM was found to vary based on: (1) chosen normalization scheme, (2) the stage within the task, (3) the object grasped, and (4) the trajectory of the motion. The RCM addresses some of the limitations of current measures of coordination because it is applicable to discrete motions, does not rely on cyclic repetition, and uses velocity-based measures. Future work will explore clinically relevant differences in the RCM as it is expanded to evaluate different tasks and patient populations.     

Quantification of protein A-gold staining for peroxisomal enzymes by confocal laser scanning microscopy.

The protein A-gold technique has been widely applied for visual localization and quantification of various antigens by electron microscopy. Observation of specimens stained by the protein A-gold technique with conventional light microscopy is difficult because of insufficient sensitivity of the staining. Light microscopic visualization and quantification of the reaction products were attempted employing a confocal laser scanning microscope (CLSM). Liver tissues of normal and peroxisome proliferator-treated rats were fixed and embedded in Lowicryl K4M resin. Ultrathin and thin sections were stained for catalase and a peroxisome-specific beta-oxidation enzyme by the protein A-gold technique. Ultrathin sections were observed by electron microscopy and the labeling density for each enzyme was analyzed with an image analyzer. Thin sections were observed with a CLSM in the reflection mode and the intensity of the light reflection was analyzed under the same conditions for all specimens. A comparison of these two observation procedures was also attempted using liver tissues stained with various concentrations of the antibody for catalase. The intensity of the reflection for each, as observed by CLSM, correlated well with the labeling density observed by electron microscopy. CLSM made it possible to quantify and to directly observe protein A-gold staining at the light microscopic level.(J Histochem Cytochem 47:1343-1349, 1999)     

Tissue-type plasminogen activator in rat adrenal medulla

Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenaline. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Tissue-type plasminogen activator in rat adrenal medulla

Summary Rat adrenal glands were stained immunocytochemically using antibodies against plasminogen activators of the tissue-type (t-PA) and urokinase-type (u-PA). A subpopulation of the cells in the adrenal medulla showed intense cytoplasmic t-PA immunoreactivity, while no u-PA immunoreactivity was detected in any adrenal cells. Fluorescence microscopy of adjacent sections demonstrated that the cells stained for t-PA contained noradrenalin. Analysis with a histochemical fibrin slide technique demonstrated a plasminogen-dependent fibrinolysis in the adrenal medulla. SDS-PAGE of adrenal gland extracts followed by zymography established the molecular weight of this plasminogen activator to be similar to that of rat t-PA. In addition SDS-PAGE followed by immunoblotting with anti-t-PA IgG of adrenal gland extracts revealed one band with an electrophoretic mobility indistinguishable from that found in the zymography. When tissue-sections and immunoblots were incubated with antibodies absorbed with highly purified t-PA no staining was found. In view of the previous finding of t-PA in growth hormone-containing cells of the pituitary gland, these findings substantiate that t-PA can be found in the intact normal organism outside endothelial cells, and further point to t-PA having a function in endocrine cells.     

Reflection contrast microscopy of ultrathin sections in immunocytochemical localization studies: a versatile technique bridging electron microscopy with light microscopy

Reflection contrast microscopy (RCM) of ultrathin sections was recently introduced as a sensitive technique for visualization with enhanced definition in immunogold histochemistry. Experience of using RCM as a major tool in immunocytochemical research in different fields is summarized, e.g. oncology, nephrology and embryology. The sensitive visualization of immunocytochemical labels, gold particles or peroxidase-diaminobenzidine deposits in or on ultrathin sections, by RCM instead of electron microscopy is demonstrated. RCM of ultrathin sections is an adequate light microscopical alternative for immunoelectron microscopy, since an overview of both label and tissue is obtained with a high image definition and high contrast of label. In the studies presented, RCM is shown to provide a better gradation in staining intensity and staining pattern than other light microscopical methods. Moreover, a precise localization of multiple labels is obtained with this method. Besides the applications shown, ultrathin section visualization by RCM is very useful for correlative light- and electron microscopical studies of fine structures. Commercially available fluorescence microscopes can be adapted for proper RCM functioning; an adaptation scheme and list of microscopes tested is provided.     

Local cell membrane deformations due to receptor-ligand bonding as seen by reflection microscopy

Understanding surface receptor clustering and redistribution processes at the cell-matrix contact zone requires detailed knowledge of the spatial integration of these molecules in the architecture of this complex interface. Here we present and discuss critically a procedure to extract such information combining reflection contrast microscopy (RCM) and reflection interference microscopy (RIM). As model system, we used living human umbilical vein endothelial cells (HUVEC) adhering to laminin-coated surfaces and investigated the distribution of the alpha2beta1 (CD29/CD49b) integrin at the contact zone of these cells. First, we applied freeze-fracture electron microscopy to gain information on microscopic details of the alpha2beta1 distribution at the contact zone. Next, we visualized and analyzed the overall lateral distribution of the integrins applying RCM using immunogold-labeling with 10 nm labels and a special silver enhancement technique. We found that RCM can be used to determine the lateral position of the marked receptor molecules to an accuracy of about 100-200 nm, instead of large morphological changes at the contact zone during silver enhancement. Finally, we combined RCM with RIM and analyzed the interference pattern of the contact zone around the label positions. Thus, we were able to detect changes of the average shape of the cell membrane due to receptor-ligand bonding of a size down to the resolution of the techniques.     

On the histochemical differences of aldehyd-fuchsin positive material in the fibres of the hypothalamo-hypophyseal tract of Rana temporaria

Summary Using a modified Alcian blue/Alcian yellow method, differences in stainability in the fibres of the preoptic-hypophyseal tract of Rana temporaria were observed. Near the preoptic nucleus most of the fibres stained yellow, in the tract the fibres stained lightgreen or showed green inclusions on a yellow background, whereas at the fibre-endings a colour switch to blue-green was found. This colour switch was the most pronounced in the neural lobe, where the neurosecretion stained blue-green, whereas in the fibre-endings in the outer zone of the median eminence a colour switch to green was noticed.The results can best be explained with the assumption that a modification or transformation of the neurosecretion occurs during its transport from the preoptic nucleus to the fibre-endings.Literature related to this hypothesis is discussed.     

Response of red cell and plasma volume to prolonged training in humans

To clarify the role of progressive heavy training on vascular volumes and hematologic status, seven untrained males [maximal O2 uptake (VO2max) = 45.1 +/- 1.1 (SE) ml.kg-1.min-1] cycled 2 h/day at an estimated 62% of VO2max. Training was conducted five to six times per week for approximately 8 wk. During this time, VO2max increased (P less than 0.05) by 17.2%. Plasma volume (PV) measured by 125I increased (P less than 0.05) from 3,068 +/- 104 ml at 0 wk to 3,490 +/- 126 ml at 4 wk and then plateaued during the remaining four wk (3,362 +/- 113 ml). Red cell (RBC) mass (RCM) measured by 51Cr-labeled RBC did not change during the initial 4 wk of training (2,247 +/- 66 vs. 2,309 +/- 128 ml). As well, no apparent change occurred in RCM during the final 4 wk of training when RCM was estimated using PV and hematocrit (Hct). Collectively, PV plus RCM, expressed as total blood volume (TBV), increased (P less than 0.05) by 10% at 4 wk and then stabilized for the final 4 wk. During the initial phase of training, reductions (P less than 0.05) were also noted in Hct (4.6%), hemoglobin (Hb, 4.0%), and RBC count (6.3%). In contrast, an increase in mean cell volume (MCV, 1.7%) and mean cell Hb (2.3%) was observed (P less than 0.05). From 4 to 8 wk, no further changes (P greater than 0.05) in Hb, RBC, and MCV were found, whereas both mean cell Hb and Hct returned to pretraining levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron-microscopic study of the secretion of the ependymal cells in the domestic cat (ependymin-beta cells).

We have studied, by electron microscopy, the ultrastructural aspects of secretion (neurosecretion) of the ependyma of the third ventricle of the domestic cat. We have found cytoplasmic protrusions and isolated masses of cytoplasm, some with homogeneous cytoplasm and others with very dense granulation (protein-beta?). Axons, synaptic terminals and free secretory granules in the ventricular lumen were also seen. The existence of ependymin-beta cells (ependymocyte-beta) and axohormonal buttons is suggested. The ependymal cells are classified into seven types: (1) covering ependymocytes, (2) tanycyt ependymocytes, (3) secretory ependymocytes, (4) ependymocytes-beta, (5) neurosecretory ependymocytes, (6) neurosensorial ependymocytes (crown-like) and (7) supraependymal microgial ependymocytes. A neurohormonal hypothesis and the possible existence of one or more cerebral hormones (neurohormones) are suggested. These hormones would flow into the CSF through some of the ependymal cells (by microapocrine secretion, liberation of neurosecretion granules, or by axohormonal buttons): this could be the most important link in the endocrine system, assuring the functional unity throughout the ventricular system of the cerebrospinal axis which it winds through, although its basic influence is exercised) on the hypophysis level as a vertex of the classical endocrine system.     

Effect of x-ray contrast substances on the complement system of rats

The purpose of the work was to demonstrate the effect of radiographic contrast media (RCM) 50% bilignost, 76% triombrast, 80% iodamide-380 on the complement system in rats. It has been found that RCM cause alternative complement activation during incubation with the whole blood or serum in vivo and in vitro. The RCM effect value depends on the drug dose and increase in the order: triombrast iodamide bilignost. Different sensitivity of animals to the effect of RCM on the complement system has been shown.     

Immunocytochemical Localization of Chondroitin and Chondroitin 4- and 6-Sulfates in Developing Rat Cerebellum

Monoclonal antibodies specific for unsulfated, 4-sulfated, and 6-sulfated disaccharide "stubs" that remain attached to the core protein after chondroitinase ABC digestion of chondroitin/dermatan sulfate proteoglycans have been used to study the localization of chondroitin and the two isomeric chondroitin sulfates in developing rat cerebellum. At 1-2 weeks postnatal, unsulfated chondroitin is present in the granule cell layer, molecular layer, and prospective white matter, but there was no staining of the external granule cell layer other than light staining of Bergmann glia fibers. By 3 weeks postnatal, staining of the molecular layer has disappeared and has diminished in the white matter, whereas in adult cerebellum only the granule cell layer remains stained. The staining pattern of chondroitin 4-sulfate is similar to that for chondroitin at 1-2 weeks postnatal, but in contrast to chondroitin, chondroitin 4-sulfate increases in the molecular layer at 3 weeks, and this becomes the most densely stained region of adult cerebellum. Chondroitin 6-sulfate is present predominantly in the prospective white matter of 1-2 week postnatal cerebellum, although significant staining of the granule cell layer is also seen. By 3 weeks postnatal the granule cell staining of chondroitin 6-sulfate has decreased, and in adult cerebellum staining is seen only in the white matter and to a lesser extent in the granule cell layer. Electron microscopy confirmed the presence of chondroitin sulfate in the cytoplasm of neurons and glia of adult brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reb1-1 mutation of Arabidopsis alters the morphology of trichoblasts,the expression of arabinogalactan-proteins and the organization of cortical microtubules

The root epidermal bulger 1 ( reb1) mutant of Arabidopsis thaliana (L.) Heynh. is characterized by a reduced elongation rate of the primary root and by the bulging of many, but not all, root epidermal cells. In this study, we investigated cell wall structure of root epidermal cells in reb1-1 by using serial sectioning, and light and electron microscopy in combination with immuno-cytochemistry and polysaccharide staining. We found that: (i) Cell bulging in the mutant was initiated in the zone of elongation of the root, and occurred exclusively in trichoblasts. (ii) reb1-1 and wild-type root cells stained identically with anti-pectin antibodies, such as JIM5. In contrast, the anti-arabinogalactan-protein antibodies, JIM14 and LM2, stained all epidermal cells in the wild type and trichoblasts preferentially, but in reb1-1 they stained the atrichoblasts only. (iii) Compared to the wild type, mutant trichoblasts had a thinner outer epidermal cell wall, which presented abnormal periodic acid-thio carbohydrazide silver proteinate (PATAg) staining. In addition, we investigated the organization of cortical microtubules in a reb1-1 mutant line expressing a green-fluorescent protein fused to a microtubule-binding domain from human microtubule-associated protein 4. Microtubules in the swollen trichoblasts of reb1-1 were either disordered or absent entirely. Together our findings indicate that the reb1-1 mutation results in an abnormal trichoblast cell wall, and suggest that cell surface arabinogalactan-proteins are required for anisotropic expansion and for orienting cortical microtubules.     

Basic Fuchsin-Tannic Acid; A One-Solution Stain for Spore Walls in Fungi

Spore walls in fungi are stained with the supernatant obtained by centrifuging at 2000 rev/min for 15 min, equal parts by volume of 10% aqueous tannic acid and 1% aqueous basic fuchsin. Advantages of this technique are: (1) the spore walls show up better than when stained with any other dye; (2) in photomicrographs, the spore walls contrast very well with the cytoplasm; (3) it is performed quickly and easily. A comprehensive review of work done to date by other workers reveals that all of the cell wall stains found in the literature stain the spore walls faintly, or both walls and cytoplasm heavily, or only the cytoplasm.     

Influence of ionic and non-ionic radiographic contrast media on leukocyte adhesion molecules

BACKGROUND: Many papers have focused on the importance of granulocytes in the process of reperfusion and ischemia. Most of the clinical studies measured several parameters of this process during and after coronary angiography, without taking into account the effect of the radiographic contrast media (RCM) used during this procedure. MATERIALS AND METHODS: We performed a randomized patient study (n = 37) to evaluate the effect of ionic and non-ionic RCM on granulocyte adhesion during coronary angiography. We also evaluated the influence of the ionicity and osmolarity of the different substances on granulocyte adhesion molecules in in vitro experiments. RESULTS: The osmolarity of patient serum samples increased from 302 +/- 1 to 309 +/- 1 mOsm/kg (p < 0.05) after infusion of RCM. The CD11b expression in the samples of the non-ionic RCM treated group increased from 221 +/- 21 MFI to 377 +/- 30 MFI (p < 0.05) measured as the absolute mean fluorescence intensity (MFI), yet did not alter significantly in the ionic RCM group. In contrast, the in vitro experiments showed a reduction of the CD11b expression from 360 +/- 70 MFI to 149 +/- 30 MFI (p < 0.05) in the ionic RCM group. CONCLUSIONS: The upregulation of adhesion molecules was significantly reduced in vivo with ionic RCM, while ionic substances caused opposite effects in vitro. This effect should be taken into account when performing leukocyte functional analysis of samples taken during angiography.