Deparaffination time: a crucial point in histochemical detection of tissue copper

The search for a sensitive histochemical method for revealing tissue copper has been the object of many workers in the past. In spite of multiple methods available, the occurrence in clinical practice of negative histochemical stains, even in cases with high copper levels demonstrated by quantitative methods is very high.This study was aimed at verifying the role of technical variations in the sensitivity of the Timm method and, in particular, the role of the dewaxing time of paraffin sections. To this end, 15 liver specimens, 10 from patients affected by Wilson's disease and 5 newborn livers were fixed in 10% formalin, paraffin embedded and routinaly processed. Four 4-micron sections from each case were rinsed in xylene for 10, 20, 60 min, and for 24 hrs. All sections were stained with Timm's method. In 13 out of the 15 liver biopsies utilized in this study, the sensitivity of Timm's method in revealing copper deposits in liver cells appeared to be dependent on the dewaxing time. In two other cases, reactivity of copper granules to Timm solution did not change significantly with the different deparaffination times. The best results were obtained by rinsing sections in xylene for 24 hrs, the worst in sections treated with xylen for 10 minutes. In particular, in five cases of Wilson's disease, Timm stain applied to sections following ten minutes of xylene were completely negative, while copper granules were clearly evidenced in the same section following an overnight bath in xylene. Our data show that an overnight bath of paraffin sections in xylene may completely change the sensitivity of Timm stain in revealing copper deposits in the liver, relaunching copper histochemistry in the diagnosis of copper-related liver diseases.     

Pneumocystis carinii in corticosteroid-treated voles: a comparison of three different staining methods.

Pneumocystis carinii (PC) is an opportunistic pathogen which causes clinical disease in immunocompromised hosts. Three different staining protocols were employed to detect this organism in lung samples of corticosteroid treated voles in order to discover a suitable method for large-scale screening. The procedures employed were: Grocotts methenamine silver (GMS)-stained paraffin sections, toluidine blue O-stained impression smears, and methenamine-silver-stained frozen sections. GMS-stained paraffin sections were relatively easy to interpret and gave more positive results than the other methods. It seemed to be the satisfactory method for large-scale population analyses. An unexpected result was that methylprednisolone treatment did not induce in voles a similarly fatal pneumocystosis infection as occurred in rats. All infections found in voles were mild. This might be due to species-dependent differences in metabolizing methylprednisolone.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

A Method for Histological Examination of Undecalcified Teeth

A method is presented for histological examination of undecalcified ground sections of tooth roots affected with periodontal disease. The roots were placed in Karnovsky's fixative overnight, postfixed in 2% buffered osmic acid, and dehydrated in an ascending series of ethanol. The specimens were then infiltrated with propylene-oxide and Epon-Araldite resin, embedded in Epon-Araldite, and sections were prepared using a cutting and grinding system. The resulting ground sections were 8-12 μm thick. The sections were allowed to air dry at room temperature. When thoroughly dried, a coverglass was applied using resinous mounting medium DPX. The specimens were examined by phase-contrast microscopy. The method is useful for simultaneous examination of mineralized dental tissue and bacterial morphotypes covering the root surface of teeth involved with periodontal disease.     

Identification of cell wall deficient forms of M. avium subsp. paratuberculosis in paraffin embedded tissues from animals with Johne's disease by in situ hybridization

M. avium subsp. paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease (JD) in ruminants leading to enormous economical losses in dairy and meat industries worldwide. During the subclinical stage of the disease, the infected animals are difficult if not impossible to detect by the available diagnostic tests including the PCR based ones. Although only considered an animal pathogen, cell wall deficient (CWD) forms of M. paratuberculosis have been isolated from patients with sarcoidosis and Crohn's disease (idiopathic diseases) in humans. Hence, the CWD form of this organism has been suspected to play a role in the pathogenesis of these diseases by persisting in the affected tissues and triggering a localized immune response and pathology. Differentiating between the CWD and acid-fast forms of this organism may lead to the determination of whether the CWD form is the pathogenic form in the subclinical cases of JD in animals and/or the etiologic agent for the above human diseases. To localize such organisms in tissue sections, CWD forms of mycobacteria were prepared in vitro and injected into beef cubes which were then formalin fixed and paraffin embedded. An in situ hybridization (ISH) technique, combined with the IS900 M. paratuberculosis-specific probe labeled with digoxigenin, was developed for the detection of nucleic acids specifically from the CWD forms but not their acid-fast forms in tissue sections. Specificity was confirmed by the negative finding with an irrelevant probe and with control tissue preparations containing CWD cells of related mycobacteria and unrelated organisms. This ISH procedure provides a way to distinguish between the acid-fast and CWD forms of M. paratuberculosis and to localize them in tissue sections. ISH may prove useful to evaluate the significance of CWD forms of M. paratuberculosis in the pathogenesis of JD, Crohn's disease and sarcoidosis.     

iNOS activity in the aged rat liver tissue

Free radical damage to many cellular components has been proposed as the main mechanism underlying the aging process. In the liver, NO can be generated by iNOS, but also by the constitutively expressed endothelial NOS (eNOS). iNOS enzyme appears to be expressed in liver disease such as cirrhosis and fulminant hepatitis, while the eNOS is expressed in physiological conditions. Ten young and ten old Wistar rats were sacrificed and their livers were excised. Liver sections were incubated with an anti-iNOS antibody of rabbit origin. RT-PCR and Western blot analysis were performed and nitric oxide activity was calculated. A significant increase of iNOS immunoreactivity was seen in the aged liver sections versus young liver sections. iNOS protein is expressed in greater quantities in the aged group, compared to the young group. In this study we show, for the first time, that aging in the rat liver is accompanied by a spontaneous induction of iNOS mRNA, high levels of iNOS protein and immunohistochemistry/image analysis.     

利用偏光影像技术定量观察大鼠胸主动脉中纤维结构

目的：本实验选取大鼠胸主动脉为研究对象,探讨定量偏光影像技术对双折光性物质的成像特点与适用研究领域。方法：采用Abrio液晶偏光影像系统在未经染色条件下对青年和衰老大鼠胸主动脉纤维结构进行观察,然后对主动脉切片进行苦味酸天狼猩红染色,对比Abrio和传统偏振光技术成像的差别。结果：较传统偏振光显微镜不同,Abfio可以对纤维双折射的方位角和光程差进行定量分析。在未经染色的条件下,Abrio可对大鼠胸主动脉中具有双折光性的纤维结构清晰成像,而传统偏光显微镜在未经染色条件下不能给出有意义的信息。经苦味酸天狼猩红染色后,对比传统偏振光技术,Abrio使成像不受纤维排列方向和人为因素的影响,较为完整地反映主动脉中纤维的结构及分布情况。结论：Abrio液晶偏光影像技术能够实现对样本双折光的强弱及方向的定性和定量分析,在未经染色的条件下给出微观结构信息,适合于心血管疾病的检测与评估,可拓展应用面宽,将在未来生物医药领域中体现更大的价值。     

Optimization of a histopathological biomarker for sphingomyelin accumulation in acid sphingomyelinase deficiency

Niemann-Pick disease (types A and B), or acid sphingomyelinase deficiency, is an inherited deficiency of acid sphingomyelinase, resulting in intralysosomal accumulation of sphingomyelin in cells throughout the body, particularly within those of the reticuloendothelial system. These cellular changes result in hepatosplenomegaly and pulmonary infiltrates in humans. A knockout mouse model mimics many elements of human ASMD and is useful for studying disease histopathology. However, traditional formalin-fixation and paraffin embedding of ASMD tissues dissolves sphingomyelin, resulting in tissues with a foamy cell appearance, making quantitative analysis of the substrate difficult. To optimize substrate fixation and staining, a modified osmium tetroxide and potassium dichromate postfixation method was developed to preserve sphingomyelin in epon-araldite embedded tissue and pulmonary cytology specimens. After processing, semi-thin sections were incubated with tannic acid solution followed by staining with toluidine blue/borax. This modified method provides excellent preservation and staining contrast of sphingomyelin with other cell structures. The resulting high-resolution light microscopy sections permit digital quantification of sphingomyelin in light microscopic fields. A lysenin affinity stain for sphingomyelin was also developed for use on these semi-thin epon sections. Finally, ultrathin serial sections can be cut from these same tissue blocks and stained for ultrastructural examination by electron microscopy.     

Mycoplasma-Like organisms associated with stunting ofGypsophila paniculata L.

A new undescribed yellows-type disease ofGypsophila paniculata L. was found. Since a stunted growth is the predominant symptom the disease has been termed stunting. Electron micrographs of ultrathin sections revealed numerous mycoplasma-like organisms in diseased phloem tissues. Attempts to maintain the diseasedGypsophila plants for further investigation of this disease failed for the present in spite of all applied modifications in nutrient media.     

Prognostic significance of the nuclear DNA content in localized prostatic adenocarcinoma.

The objective of this study was to determine if the nuclear DNA content could predict disease progression in patients with stage A or B prostatic cancer. The nuclear DNA content was determined by image analysis using Feulgen-stained nuclei in tissue sections of prostatic needle biopsies from 44 patients. The patients were followed for a mean of 69.5 months, during which 12 (17%) progressed to stage D2 disease (bone or soft tissue metastases). The average times to progression to stage D2 disease were 68 months for patients who initially had stage A2 disease, 47 months for stage B1 patients and 29 months for stage B2 patients. The DNA pattern was judged diploid or normal-range (Auer type I or II histogram) in 35 tumors (80%) and aneuploid (Auer type III or IV histogram) in 9 tumors (20%). Eight (89%) of 9 tumors with an aneuploid DNA pattern and 4 (11%) of 35 tumors with a normal-range or diploid DNA pattern progressed to stage D2 disease.     

Correlation between interleukin-10 and in situ necrosis and fibrosis suggests a role for interleukin-10 in the resolution of the granulomatous response of tuberculous pleurisy patients

In order to identify mediators involved in immune-mediated disease regression, the pleural cytokine and histopathological profile was evaluated in tuberculous pleurisy patients with varied disease duration and clinical presentation, previous to chemotherapy. Interleukin (IL)-10, interferon (IFN)-gamma and tumor necrosis factor (TNF) levels in pleural fluids were shown to decrease with disease time. IL-10 was positively correlated with IFN-gamma, TNF and necrosis area in pleural sections. To parallel these findings with disease regression, individuals showing fever, anorexia, and progressive exudate in the pleural cavity (active disease) were compared with patients without symptoms and with a decrease in exudate volume (regressive disease). IFN-gamma and TNF levels were lower in regressive disease, as well as reduced necrosis area in pleural sections. Our results indicate that tissue destruction and a prominent Th1 response mark the early phase of tuberculous pleurisy and suggest that down-modulation of this response, with the possible participation of IL-10, is associated with disease resolution.     

Diagnosis of calcium pyrophosphate dihydrate deposition disease by fine needle aspiration biopsy: a case report

BACKGROUND: Calcium pyrophosphate dihydrate deposition disease is a relatively rare disease with variable clinical presentations. CASE: A 73-year-old man presented with worsening lower back pain and fever. Fine needle aspiration biopsy of the lumbar vertebral bodies (L3-L4) revealed abundant neutrophils admixed with small, birefringent, rhomboid crystals in Diff-Quik-stained smears. These crystals were confirmed as calcium pyrophosphate dihydrate on cell block sections. A diagnosis of osteomyelitis and calcium pyrophosphate dihydrate deposition disease was rendered. The patient was treated with antibiotics and responded well. CONCLUSION: Calcium pyrophosphate dihydrate deposition disease can be diagnosed by fine needle aspiration biopsy, and an accurate diagnosis can be greatly facilitated by cell block sections. However, such a diagnosis may be neglected if the specimen is not carefully inspected.     

保持培养细胞原位、原形的超薄切片制备法

本文介绍先用环氧树脂Epon812包埋剂制成丘状膜,再在膜上培养细胞,直接将膜与细胞一起包埋,制备超薄切片。此种方法不仅能克服以往培养细胞需要离心成团,容易造成细胞破碎、变形的缺点;还能避免琼脂预包埋法悬浮细胞造成的抗原封闭;并能得到为数较多的连续超薄切片。它操作简便,较好地保持了培养细胞生长的原来位置及原有形状,为培养细胞进行免疫电镜的操作和提高制备培养细胞超薄切片的数量和质量提供了一种有效的途径。     

Imaging and spatial distribution of beta-amyloid peptide and metal ions in Alzheimer's plaques by laser ablation-inductively coupled plasma-mass spectrometry

Laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) has been developed as a new strategy for detection and imaging of β-amyloid protein in immunohistochemical sections from the brains of a transgenic mouse model of Alzheimer’s disease. The distribution of β-amyloid deposits in tissue was based on measurement of Eu- and Ni-coupled antibodies. The laser-based methodologies (spot ablation, single line raster, and two-dimensional imaging) were also used to detect and map trace element distributions and thus provide a novel probe for both elemental and protein data. We also report the combination of laser capture microdissection with LA-ICP-MS as an alternative strategy for microanalysis of immunohistochemical sections.     

Immunohistology on formol-sublimate fixed and paraplast embedded kidney tissue with comparison to formalin fixation and to pre-embedding immunofluorescent staining

Many alternative methods for immunopathological evaluation of kidney tissue are now available. Immunofluorescent or immunoperoxidase staining of kidney can be performed after formalin fixation and paraffin embedding. This is also possible after fixation with formol-sublimate (Stieve's fluid) using the immunoperoxidase technique or by immunofluorescence after removal of mercury. Reduction of strong nonspecific fluorescence caused by the mercury fixative parallels the elimination of mercury as verified by X-ray microanalysis of the sections. Using a mouse model with injection of graded dilutions of antiglomerular basement membrane antibodies, immunofluorescent staining after Stieve fixation and embedding in Paraplast was about 60% of that in cryostat sections. Immunofluorescent staining after mercury removal can be followed by silver staining for detailed morphologic study of the same 1 micron Paraplast sections. A case of antiglomerular basement membrane glomerulonephritis is illustrated in more detail to show the necessity of alternative methods, including the technique presented, pre-embedding immunofluorescent staining of Epon sections, and electron microscopy, to make a reliable diagnosis of this disease.     

Chagasic infection in blood donors from hospitals in endemic regions of Chile (1982-1987). Epidemiological impact of the problem]

Chagas' disease, produced by Trypanosoma cruzi and transmitted by hematophagous triatomine bugs, exists in the Western Hemisphere from the south-western United States to central Chile and Argentina. It exists in rural and periurban sections of the northern half of Chile, with a prevalence of 16.9%. Constant rural-urban migrations have contributed to its spreading to urban sections. In order to investigate the impact of these migrations on the population susceptible of being blood donors and the probable increasing of the risk of T. cruzi transmission by blood transfusion, epidemiological surveys were carried out in donors from 22 hospitals located in the northern half of Chile. By means of an indirect hemagglutination test for Chagas' disease 16,841 blood donors were examined, arising a 2.7% of positivity, percentage that permitted to estimate that 126,477 potential blood donors infected with T. cruzi should be in the urban sections studied. These facts strengthen the need that serology for Chagas' disease must be routinely performed in endemic regions of the country, to adopt or reinforce the pertinent preventive measures.     

A quick microwave histochemical stain for copper

A rapid microwave method is described for staining copper in liver. This procedure was compared with a conventional method for copper. To this end, liver sections obtained from patients affected by several liver diseases associated with copper overload, were stained both with the standard rubeanic acid method for copper and with our modification of the same method, incorporating microwave treatment. Liver sections from a normal human newborn were used as a positive control. In Wilson's disease in the cirrhotic stage, copper was detected by the conventional method solely in periportal cells; following the microwave treatment, we were able to demonstrate copper in the whole lobule. In alcoholic cirrhosis, rubeanic acid stained copper only in a few periportal cells, while, by our modified method, copper was detected in almost all periportal hepatocytes. In chronic biliary tract disease, and in the newborn liver, copper was demonstrated in a few periportal cells by both the two histochemical procedures. In conclusion, although copper was detected by both procedures, a different degree of positivity was sometimes observed by using microwaves. Moreover, the microwave-treated sections showed more contrast and less artifacts. From a practical point of view, for the simplicity of employment and, above all, for its quickness (10 min), we suggest the use of our method in all conditions where copper overload is suspected.     

Amino Acid Residues 226–240 of τ Which Encompass the First Lys-Ser-Pro Site of τ, Are Partially Phosphorylated in Alzheimer Paired Helical Filament-τ

Abstract: A synthetic peptide corresponding to residues 226–240 (E9 peptide) of human τ, which contains an Lys-Ser-Pro motif, was used to raise a polyclonal antibody. The antibody, E9, was 10-fold less reactive with phospho-E9 peptide than with native E9 peptide. E9 antibody was used to study the extent of phosphorylation in a modified form of τ (PHF-τ) that is found in Alzheimer's disease brain and is incorporated into paired helical filaments (PHFs). E9 immunolabeled Alzheimer's disease neurofibrillary tangles and abnormal neurites in brain sections intensely, with increased immunoreactivity detected after pretreatment of sections with phosphatase. On immunoblots and ELISA, E9 reacted with PHF-τ and recombinant human τ but not with the high and middle molecular weight neurofilament proteins. Phosphatase treatment of PHF-τ improved the E9 immunoreactivity by 30–50%. Dephosphorylated high but not middle molecular weight neurofilament protein became reactive with E9. These results indicate that <50% of the PHF-T is phosphorylated in the subregion corresponding to residues 226–240 of τ and suggest that the phosphorylation of this region may not be essential for PHF formation.     

Immunolocalization of influenza A virus and markers of inflammation in the human Parkinson's disease brain

Although much is known regarding the molecular mechanisms leading to neuronal cell loss in Parkinson's disease (PD), the initiating event has not been identified. Prevailing theories including a chemical insult or infectious agent have been postulated as possible triggers, leading to neuroinflammation. We present immunohistochemical data indicating the presence of influenza A virus within the substantia nigra pars compacta (SNpc) from postmortem PD brain sections. Influenza A virus labeling was identified within neuromelanin granules as well as on tissue macrophages in the SNpc. Further supporting a role for neuroinflammation in PD was the identification of T-lymphocytes that colocalized with an antibody to caspase-cleaved Beclin-1 within the SNpc. The presence of influenza A virus together with macrophages and T-lymphocytes may contribute to the neuroinflammation associated with this disease.