Pretreated Sections Research Articles

Susceptibility of pretreated wood sections of Norway spruce (Picea abies) clones to enzymatic hydrolysis

The structure of softwoods, which confers resistance to degradation through hydrolysis and decay, currently limits their use for the production of biofuels. However, since wood is very heterogeneous, it is possible that differences in wood properties within and between trees could differentially affect its processability. In this research, heartwood (inner) and sapwood (outer) from Norway spruce ( Picea abies (L.) Karst.) clones were enzymatically hydrolyzed by Trichoderma viride cellulases after concentrated acid pretreatment. Wood sections with two particle sizes were compared based on their susceptibility to enzymatic hydrolysis, evaluated by assaying the formation of hydrolysis products and measured as reducing sugar yield (RSY). We also studied the relationship between RSY and the susceptibility to Heterobasidion parviporum wood decay and whether these traits are reflected in wood density and yield. Wood from the outer section produced more RSY with higher glucan but lower lignin content than wood from the inner section. Furthermore, susceptibility to enzymatic hydrolysis was positively correlated with H. parviporum wood decay, while both processes were negatively correlated with wood density. Our results revealed the importance of clonal trials for identifying suitable lignocellulosic biomass when considering wood properties and indicate that potential genotypes for the production of biofuels are not necessarily the most productive.

Distribution of anticancer antibiotic daunomycin in the rat heart and kidney revealed by immunocytochemistry using monoclonal antibodies

Two monoclonal antibodies (ADM-1-11 and 79-31 mAbs) were raised against daunomycin (DM) conjugated to bovine serum albumin via the cross-linker N-(gamma-maleimidobutyryloxy)succinimide. The monoclonal antibodies (mAbs) specifically detected DM as well as its analogs doxorubicin and epirubicin, but did not react with other anticancer antibiotics, including pepleomycin, mitomycin C, and actinomycin D. The mAbs reacted strongly with glutaraldehyde-conjugated DM in an enzyme linked immunosorbent assay (ELISA) used as a model system for immunocytochemistry as well as in appropriately pretreated sections of tissues from animals injected with DM. No staining occurred in tissues from uninjected animals. In order to perform DM ICC a number of tissue treatment conditions critical to the detection of low molecular weight substances were employed. Uptake of DM was studied in rats after a single i.v. or i.p. administration of the drug. In the heart, accumulation of DM occurred in nuclei and in the cytoplasm. In the kidney, DM immunoreactivity accumulated in all segments of the nephron except for the proximal tubules. Since the proximal tubules are known to be where a variety of transport systems including P-glycoprotein (Pgp) and organic anion-transporting polypeptides (OATPs) in drug interactions occur, the absence of DM accumulation in these segments may reflect a transport phenomenon depending upon such transporters. The availability of methods to study sites of accumulation of DM offers possibilities for understanding toxic side effects of this drug on the heart and kidney. Moreover, the immunocytochemical methodology developed may prove useful for the localization of other low molecular weight drugs that can be fixed in situ by glutaraldehyde.

Demonstration of α-Synuclein Immunoreactivity in Neuronal and Glial Cytoplasm in Normal Human Brain Tissue Using Proteinase K and Formic Acid Pretreatment

α-Synuclein (αS), a presynaptic nerve terminal protein, is now known to be a major component of neuronal and glial cytoplasmic inclusions in α-synucleinopathies (Lewy body disease and multiple system atrophy). However, αS has not been identified in either neuronal or glial cytoplasm in formalin-fixed, paraffin-embedded tissue sections from the normal human brain. Previous studies have shown that pretreatment with either proteinase K or formic acid enhances αS immunoreactivity. The aim of the present study was, therefore, to study the effects of pretreatment with proteinase K and formic acid on αS immunoreactivity in vibratome sections of brain tissue taken from normal human subjects. In addition to presynaptic staining, αS immunostaining was recognized in neuronal perikarya in the pretreated sections; this immunoreactivity was more intense in sections taken from the deeper layers of the cerebral neocortex, the CA2/3 region of the hippocampus, and the substantia nigra. This pattern of αS expression coincides with the distribution of intraneuronal inclusions in αS transgenic animals as well as in human autopsy tissue taken from patients with Lewy body disease. Furthermore, intense immunoreactivity was also found in the cytoplasm of astrocytes and oligodendrocytes throughout the brain. These findings suggest that a significant amount of αS is also present in the neuronal and glial cytoplasm in the normal human brain.

P53 gene product expression in resected non-small cell carcinoma of the lung, with studies of concurrent cytological preparations and microwave antigen retrieval.

AIM: To document the frequency and extent of p53 gene product expression in paraffin sections of resected non-small cell carcinoma of the lung and in cytological preparations of the same...

Demonstration of CRP immunoreactivity in brains of Alzheimer's disease: immunohistochemical study using formic acid pretreatment of tissue sections

C-reactive protein (CRP) is a well-known serum protein which increases during inflammation and deposits in destructed tissues. To establish whether CRP appears in brain of Alzheimer's disease (AD), we immunohistochemically investigated tissue sections which were pretreated with formic acid. Positive immunostainings by anti-CRP antibodies were clearly recognized in senile plaques (SP) in the pretreated tissue sections whereas very weak immunostainings in non-treated sections. These findings may suggest that the formation process of SP contains an acute phase of inflammatory state.

Prevalence of A4 amyloid protein deposition in the chinese population determined by immunocytochemistry

The study was performed on 203 randomly selected autopsy brains in Chinese in Hong Kong, aged 50 years or more, dying in a regional general hospital. Histological sections of parietal blocks and hippocanpal blocks were stained with H & E and congo red. Formic acid pretreated sections were immunostained with rabbit antiserum against synthetic A4 protein (kindly supplied by Prof. Masters) using avidin- biotinylated peroxidase technique. A4 protein deposits were quantitated manually using a graticule placed on the cortical ribbon and a mean plaque score (PS) was assigned. The quantity of A4 protein deposits is strikingly greater than the amount of amyloid seen by congo red polarization microscopy. The prevalence of A4 protein deposits and the mean PS rise with age. Its deposition does not correlate well with clinical diagnosis of Alzheimer's disease. Prevalence in Chinese is statistically lower than those in Australia (Masters 1988) and in U.K. (Mann 1990). NGMASTERSMANNNumber20311060Prevalence of A4 deposits≥50 yr20.2%45%51%≥65 yr23.5%55%78%Male: Female1:21:1Not Mentioned

Immunohistochemical detection of keratin, actin and type IV collagen in serial sections of methacarn-fixed breast cancer tissues.

Correlations among the immunolocalizations of keratin, actin and type IV collagen were investigated in serial sections of methacarn-fixed, paraffin-embedded mastectomy specimens taken from infiltrating breast cancer patients. Stainings of the filaments in pretreated sections with actinase were often weaker and/or less apparent in cancer cells than in normal mammary gland cells. If at least 30% of living cancer cells were stained, the case was scored positive. Positivity for 312C8-1 specific for basal cell type keratin 14 was observed in 8 of 60 cases, while for NCL-5D3 specific for luminal cell type keratins, positivity was observed in 57 of 60 cases. Five cases positive for HHF35 specific for muscle actin were also positive for 312C8-1 and NCL-5D3. Type IV collagen in cancer foci was patchy and/or absent, but small cancer nests surrounded completely by continuous type IV collagen, termed “in situ carcinoma components” were found in 25 of 60 cases. In all the components, stainings with 312C8-1 and/or NCL5D3 were found in some cancer cells, and the former was almost always seen in a basal orientation within the components. In con- clusion, keratin 14-positive basal cells could be identified in paraffin sections, and these findings suggest that the basal cells are closely related to continuous type IV collagen and a progressive loss of the cells may promote metastasis through fragmented type IV collagen.

Brain angiogenesis: Variations in vascular basement membrane glycoprotein immunoreactivity

Changes in the distribution and quantity of laminin and fibronectin within the basement membranes of developing or regenerating CNS blood vessels were investigated using two immunocytochemical techniques. Three models of angiogenesis were studied: normal pre- and postnatal development, wound healing, and vascularization of fetal neocortical transplants placed in the adult rat brain. Although all brain vessels were stained in enzymatically pretreated immunoreacted paraffin sections, those associated with wound and transplant sites were the most intensely reactive with both antisera during the first postoperative week. When 40-μm vibratome sections of normal adult brains were immunoprocessed, only the meninges and vessels of the circumventricular organs were stained. The remainder of the brain vasculature was immunoreactive only if sections were enzymatically treated prior to immunoprocessing. In contrast, the nascent vasculature in developing brain and the regenerating vessels at wound and transplant sites were reactive to both antisera without enzymatic pretreatment of the sections. This immunoreactivity decreased by 11 days postnatal in normal animals and 4 weeks postoperative in experimental animals, coinciding with the period of astrocytic contact and complete vascular basement membrane formation in both cases. The variations in staining pattern and intensity may be reflections of differences in the quantity of laminin and fibronectin within the basement membranes of proliferating and/or non-blood-brain barrier vasculature. However, the results of the different experimental protocols suggest that immature vascular basement membranes may have a molecular configuration that does not require an enzymatic unmasking step to react with the antisera. Alternatively, the looseness of the surrounding neuropil inherent in developing and injured CNS could allow the antisera greater access to basement membrane antigens.

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.

Renal distribution of collagen types III, IV and V in various glomerular diseases

The purpose of this study is to examine the immunochemical changes of the glomerular basement membrane (GBM) and the mesangium, in pretreated paraffin-embedded sections with trypsin by utilizing monoclonal antibodies to type III (anti-III), type IV (anti-IV) and type V (anti-V) collagens. We observed 6 normal kidneys and 44 kidneys with various renal diseases. In normal human kidney the staining with anti-IV demonstrated GBM, mesangium, Bowman's BM, tubular BM and capillary BM. Anti-V was also seen in the interstitium. On the other hand, anti-III stained only interstitium. Thickened GBM in membranoproliferative glomerulonephritis (MPGN) and diabetic nephropathy, and irregular GBM in Membranous Nephropathy and Alport's syndrome were also evident in anti-IV stain, while widened mesangial area was seen in anti-V rather than anti-IV stain. In severely proliferative GN, anti-III as well as anti-IV and anti-V was detected in the mesangium in spite of existence of neither adhesion nor Bowman's gap. In MPGN type II, anti-III was observed along the GBM. In obsolescent glomeruli, anti-IV was not always detected although anti-V was constantly seen. On the other hand, anti-III was markedly positive in the crescents and obsolescent glomeruli. These results suggest that it is possible for mesangial, endothelial and epithelial cell to produce several types of collagens and type III collagen is closely related to the process of the glomerular obsolescence.

Histochemical detection of alpha-D-glucosidases and their molecular forms with 5-Br-4-Cl-3-indoxyl-alpha-D-glucoside.

5-Br-4-Cl-3-Indoxyl-alpha-D-gluco(pyrano)side was found to be the most suitable synthetic substrate for the demonstration of alpha-D-glucosidases in situ. Using an azoindoxyl procedure with hexazotized pararosaniline or new fuchsine at pH 5 in freeze-dried celloidine-mounted cryostat sections acid alpha-D-glucosidase (EC 3.2.1.20) was shown for the first time in lysosomes of many cells of fetal and adult rat, mouse, guinea-pig, marmoset and human organs. At pH 6.5, in chloroform-acetone pretreated cryostat sections plasma membrane alpha-D-glucosidases were shown in the brush border of enterocytes of the small and large intestine, in the brush border of proximal renal tubule cells and in the stereocilia of the epididymal duct. In an indigogenic procedure with ferricyanide/ferrocyanide as redox catalysator plasma membrane alpha-D-glucosidases were depicted as well as with the azo-indoxyl method; the demonstration of the acid alpha-D-glucosidase was inferior to that achieved with the azo-indoxyl procedure. Using tetrazolium salts as capture reagent intracellular localization was unsatisfactory. In enterocytes, a localization in the Golgi apparatus was shown by the azo-indoxyl procedure only. Analytical isoelectric focusing revealed organ-dependent differences of plasma membrane and lysosomal alpha-D-glucosidases. Compared with the already existing methods the azo-indoxyl and indigogenic procedures are by far the most suitable techniques.

Identification of fiber types in rat skeletal muscle based on the sensitivity of myofibrillar actomyosin ATPase to copper

Experiments are reported demonstrating that differential rates of inactivation of the histochemical staining for myofibrillar actomyosin ATPase in rat skeletal muscle fibers exist following inclusion of low concentrations of Cu2+ in the preincubation medium. This response of rat muscle occurs at near neutral (7.40), acid (4.60), and alkaline (10.30) pH. The response to Cu2+ appears to result from a binding of Cu2+ onto the myofibrillar complex, probably on myosin itself, as it can be reversed by soaking of the pretreated muscle sections in sodium cyanide or the Cu2+ chelator diethyldithiocarbamate. The pattern of modification of the staining pattern following pretreatment with Cu2+ is the mirror image of that produced by pretreatment with acid. The results demonstrate that the inclusion of Cu2+ in the preincubation media for the myofibrillar actomyosin ATPase can be a useful tool to differentiate fiber types. They also support the earlier conclusion that three distinct types of type II fibers can be identified in rat skeletal muscle based on the histochemical staining for myofibrillar actomyosin ATPase.

Study on aminopeptidase A.

A comparative biochemical and histochemical investigation of aminopeptidase A (APA, E.C. 3.4.11.7) was carried out. α-Glu-1NA, α-Glu-2NA, α-Glu-MNA and Asp-2NA were used as substrates, Fast Blue B (FBB), hexazotized new fuchsin (HNF) and hexazonium-p-rosaniline (HPR) as coupling agents. Biochemical determination of APA activity was performed in whole homogenates as well as in homogenized freeze-dried cryostat sections of many rat, guinea-pig and human organs. α-Glu-2NA proved to be the best substrate for the biochemical determination of APA activity. It displays favourable kinetic properties and abilities for spectrophotometric as well as fluorometric measurements.K m of rat kidney enzyme amounts to 0.15–0.43 mM, of rat jejunum enzyme 0.35 mM, of myocardial enzyme 0.2 mM and of rat brain enzyme 0.25 mM. The enzyme is activated by Ca2+ and inhibited by EDTA (1 mM) and 1,10-phenanthroline (1 mM). E600, DFP and PCMB (1 mM) did not influence APA activity. The activity was detected in every organ examined. Great organ and species differences were demonstrated. The highest APA activity resides in the kidney and in the small intestinal mucosa. The structural association of APA is not absolutely firm and up to 30% (depending on the organ and eventual pretreatment of sections with chloroform-acetone which impedes the leakage to some extent) escapes into the aqueous incubation solution. The highest values were recorded in phosphate and cacodylate buffers followed by citrate phosphate, citrate, Tris-HCl and acetate buffers. There were no great differences in activities measured at pH 6.5 and 7.2. For the histochemical demonstration of APA activity α-Glu-MNA is the substrate of choice.K m estimated biochemically amounts to 0.3–0.43 mM for the kidney enzyme, 0.35 mM for the rat jejunal enzyme and 0.5 mM for rat brain enzyme.K m estimated by microdensitometric measurements of the reaction product in the brush border of cells of proximal tubules and of jejunal enterocytes at the sites of villi amount to 0.6 and 0.4 mM respectively. The best results were obtained using 2–4 mM concentration of α-Glu-MNA, and satisfactory results using 0.36–0.72 mM concentration. FBB appears to be the best coupling agent in the routine. The most sensitive way to depict APA in sites with low and moderate activity is to use chloroform-acetone pretreated cryostat sections adherent to semipermeable membranes. Sites with high enzyme activity are best revealed upon incubation of chloroform-acetone pretreated cryostat sections adherent to slides. Of the unsubstituted naphthylamine derivative of α-Glu or Asp α-Glu-2NA is superior to α-Glu-1NA or Asp-2NA. In this case HPR or HNF are to be used as coupling agents in cryostat sections adherent to slides. Due to a higher decomposition rate and higher inhibitory influence of diazonium salts on APA activity in the alkaline pH range it is advisable to carry out the reaction at pH 6.5–7. The most important binding sites of APA are: The functional significance of APA is discussed in relation to the degradation of angiotensin II (endothelium, kidney) and to other brush border peptidases releasing amino acids which are absorbed (enterocytes). The histochemical demonstration is indispensable for a correct interpretation of biochemical data.

Interaction of [3H]gibberellin A1 with a sub-cellular fraction from lettuce (Lactuca sativa L.) hypocotyls

The relationship between elongation growth and the incorporation of [(3)H]gibberellin A1 ([(3)H]GA1) into a 2,000g pelletable (2KP) fraction from lettuce (Lactuca sativa L., cv. Arctic) hypocotyl sections has been examined. Sections were loaded with incremental amounts of GA1 under conditions where growth was arrested (5° C) or permitted (30° C) and, after 16 h, all were transferred to a GA-free medium at 30° C. Growth and 2KP radioactivity were measured at this point and after a further 24 h in the chase medium. Uptake was reduced by 80% at 5° C, as compared to 30° C, but 2KP labelling and protein synthesis were only reduced by half. The growth rate of the 5° C pretreated sections during the chase period was comparable to that observed during the pulse in the 30° C material but the dose/response relationship was flatter. Low temperature sections incorporated a much higher percentage of GA1 uptake into the 2KP fraction (27% at maximum) but the absolute levels of labelling at this temperature were lower than those measured at 30° C. The data are interpreted as showing that 2KP labelling is not a consequence of growth. It must either precede response or be an unconnected concurrent process.