Zamboni's Solution Research Articles

Enzyme-labeled Antigen Method: Factors Influencing the Deterioration of Antigen-binding Activity of Specific Antibodies during Formalin Fixation and Paraffin Embedding.

The enzyme-labeled antigen method is an immunohistochemical technique detecting plasma cells producing specific antibodies in tissue sections. The probe is an antigen labeled with an enzyme or biotin. This immunohistochemical technique is appliable to frozen sections of paraformaldehyde (PFA)-fixed tissues, but it has been difficult to apply it to formalin-fixed, paraffin-embedded (FFPE) sections. In the current study, factors inactivating the antibody reactivity during the process of preparing FFPE sections were investigated. Lymph nodes of rats immunized with horseradish peroxidase (HRP) or a mixture of keyhole limpet hemocyanin/ovalbumin/bovine serum albumin were employed as experimental models. Plasma cells producing specific antibodies, visualized with HRP (as an antigen with enzymatic activity) or biotinylated proteins in 4% PFA-fixed frozen sections, significantly decreased in unbuffered 10% formalin-fixed frozen sections. The positive cells were further decreased by paraffin embedding following formalin fixation. In paraffin-embedded sections fixed in precipitating fixatives such as ethanol and acetone and those prepared with the AMeX method, the antigen-binding reactivity of antibodies was preserved. Fixation in periodate-lysine-paraformaldehyde and Zamboni solution also kept the antigen-binding reactivity in paraffin to some extent. In conclusion, formalin fixation and paraffin embedding were major causes inactivating antibodies. Precipitating fixatives could retain the antigen-binding reactivity of antibodies in paraffin-embedded sections.

QUERCETIN SUPPLEMENTATION PREVENTS CHANGES IN THE SEROTONIN AND CASPASE-3 IMMUNOREACTIVE CELLS OF THE JEJUNUM OF DIABETIC RATS.

Serotonin (5-HT) is present in the epithelial enterochromaffin cells (EC), mast cells of the lamina propria and enteric neurons. The 5-HT is involved in regulating motility, secretion, gut sensation, immune system and inflammation. Evaluate the effects of diabetes and quercetin supplementation on serotoninergic cells and its cell loss by apoptosis in jejunal mucosa of streptozotocin-induced diabetic rats (STZ-rats). Twenty-four male Wistar rats were divided into four groups: normoglycemic (C), normoglycemic supplemented with 40 mg/day quercetin (Q), diabetic (D) and diabetic supplemented with 40 mg/day quercetin (DQ). After 120 days, the jejunum was collected and fixated in Zamboni's solution for 18 h. After obtaining cryosections, immunohistochemistry was performed to label 5-HT and caspase-3. Quantification of 5-HT and caspase-3 immunoreactive (IR) cells in the lamina propria, villi and crypts were performed. The diabetic condition displayed an increase of the number of 5-HT-IR cells in villi and crypts, while decreased number of these cells was observed in lamina propria in the jejunum of STZ-rats. In the diabetic animals, an increased density of apoptotic cells in epithelial villi and crypts of the jejunum was observed, whereas a decreased number of caspase-3-IR cells was observed in lamina propria. Possibly, quercetin supplementation slightly suppressed the apoptosis phenomena in the epithelial villi and crypts of the STZ-rats, however the opposite effect was observed on the 5-HT-IR cells of the lamina propria. Quercetin supplementation on healthy animals promoted few changes of serotoninergic function and apoptotic stimuli. These results suggest that quercetin supplementation mostly improved the serotonergic function affected by diabetes maybe due to antioxidant and anti-inflammatory properties of quercetin.

Caracterização das proteínas caveolinas -1 e -2 na placenta de conceptos bovinos clonados transgênicos

RESUMO:A utilização da transgenia com a proteína fluorescente verde (GFP) como marcador de células de origem fetal nas placentas de clones bovinos servirá de modelo inédito para estudo morfofisiológico e imunológico da interação materno-fetal, visto que possibilitará o seu mapeamento, diferenciando as células fetais das maternas. Tal modelo terá aplicação direta, principalmente porque estes são animais que apresentam problemas em relação ao seu desenvolvimento. Com o auxílio deste modelo, pretende-se verificar o transporte de substâncias entre a mãe e o feto via endocitose, pela imunolocalização das proteínas chamadas de caveolinas. Para tanto foram utilizados 06 bovinos clonados e 30 bovinos de inseminação artificial (IA) com idade até 90 dias de gestação, os quais tiveram seu desenvolvimento interrompido mediante abate humanitário das receptoras e ovariosalpingohisterectomia, com posterior recuperação do útero gestante. Foram coletados os placentônios e o cório. Uma parte das amostras foi recortada e fixada, por imersão, em solução de parafolmaldeído a 4% ou formoldeído a 10% em tampão fosfato de sódio (PBS) a 0,1M pH 7.4, solução de Zamboni (4% de paraformoldeído, 15% de ácido pícrico, em tampão fosfato de sódio a 0,1M pH 7.4), metacarn (60% de metanol, 30% de clorofórmio, e 10% de ácido acético glacial), para verificação da morfologia e realização de imuno-histoquímica para as proteínas caveolinas -1 e -2 (CAV -1 e CAV-2). As caveolinas -1 foram localizadas nos vilos fetais e maternos, mas sua marcação mais forte foi observada no estroma endometrial. As caveolinas -2 tiveram marcação positiva no trofoblasto e membrana córioalantoide, e, especificamente em célula trofoblástica gigante binucleada. Sendo assim, os resultados mostram que a proteína CAV-1 teve uma maior expressão em relação à proteína CAV-2 e que as proteínas CAV-1 e -2 são parte da composição das cavéolas, sendo estruturas importantes e relacionadas com a transferência de moléculas para o feto, realizando a nutrição do mesmo mediante endocitose e pinocitose.

Light Microscopic and Immunohistochemical Study Revealed Sex-Dependent Kidney Morphology in DBA/2crSlc Mice

Aim: Sex-dependent differences in kidney histology have been observed in different species of the laboratory animals. The present study was conducted to evaluate the sex and strain-dependent changes in DBA/2CrSlc mouse kidney morphology by using light microscopy and immunohistochemistry. Methods: A total of 12 DBA/2CrSlc male and female mice of 2 months of age were used in this study. Mice were sacrificed by exsanguination under anesthesia using a mixture of Ketamine and Medetomidine. Both right and left kidney s were removed aseptically and central slices including hilum were cut perpendicular to the long axis of the organ and preserved in Zamboni solution. Paraffin blocks were made and tissue sections were stained with hematoxylin and eosin, and PAS stains to o bserve the general morphology of the kidney glomerulus. Immunohistochemistry was performed to detect renin positive

Mucosal Nerve Fiber Density Studies May Yield Insights into the Effects of Gastric Electrical Stimulation on Treatment Outcomes

Purpose: Debilitating nausea and vomiting frequently lead to reduced activity and quality of life in patients with gastroparesis (GP). Mucosal nerve fiber (MNF) density studies may help in characterizing Gp symptoms. Methods: Gastric mucosal tissues from 5 Gp patients (mean age 53.5 yrs; 80% Caucasian, 80% female; 1 diabetic, 2 idiopathic, 2 postsurgical Gp) and 3 age-matched controls were compared. For Gp patients, full thickness stomach biopsies, obtained at permanent stimulator implantation, were processed, and samples fixed in Zamboni's solution. Superficial MNF length density (μm/μm3) was evaluated using PGP 9.5 immunostained sections, imaged by confocal microscopy and quantified with AutoNeuron®. Results were analyzed by linear regression models using STATA 11. Gastroparesis symptom were assessed using a patient-reported, Likert-scale tool (0 to 4, none to severe). Clinical histories were studied to record duration of disease. Results: MNF density was reduced in GP, at 0.0018, compared to age-matched controls, at 0.0024 (p = 0.02). Significant associations were seen between MNF density and baseline Gp symptoms of bloating anorexia, abdominal pain, and total symptoms score (See Table). A significant association was also observed between duration of disease and reduction in MNF densities, at 6.03 [2.42, 9.64] p = 0.013.Table: Table. Associations between mucosal nerve fiber density and gastroparesis symptomsConclusion: The associations between reduced MNF density and both Gp duration and symptom severity are important findings that may improve our understanding of the natural progression of gastroparesis. The relationship between MNF density and symptom severity in particular should be explored to improve treatment and outcomes for patients with gastroparesis. Disclosure: Dr Thomas L Abell is licensor, instructor and consultyant for Medtronic Inc.

Role of Blood- and Tissue-Associated Inducible Nitric-Oxide Synthase in Colonic Inflammation

There is evidence that inducible nitric-oxide synthase (iNOS)-derived NO contributes to the pathophysiology of intestinal inflammation. The aims of this study were to assess the role of iNOS in the development of dextran sodium sulfate (DSS)-induced colonic inflammation and to define the contribution of tissue-specific iNOS expression to this inflammatory response. Study groups included: 1) wild-type (WT) mice; 2) WT=>WT bone marrow chimeras with normal iNOS function; 3) WT=>iNOS-/- chimeras (with functional blood cell iNOS, but iNOS-deficient tissue); 4) iNOS-/-=>WT chimeras (with iNOS-deficient blood cells, but normal tissue iNOS activity); and 5) iNOS-deficient mice. In WT mice and WT=>WT chimeras, DSS-induced colonic inflammation was characterized by bloody diarrhea and a high disease activity index. However, WT=>iNOS-/- and iNOS-/-=>WT chimeras and iNOS-/- mice exhibited an attenuated disease activity index, with parallel changes in histopathology. Colonic myeloperoxidase (MPO) was comparably elevated in DSS-treated WT mice (30.1+/-1.7) and WT=>WT chimeras (29.0+/-1), whereas MPO was significantly reduced in iNOS-/- mice and iNOS-/-=>WT chimeras (9.5+/-1.7 and 15.6+/-2.2, respectively). WT=>iNOS-/- chimeras exhibited the lowest MPO activity (3.7+/-0.6). Our findings implicate both blood cell- and tissue-derived iNOS in DSS-induced colonic inflammation, with tissue-associated iNOS making a larger contribution to the recruitment of inflammatory cells.

EFNS guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathy

Skin biopsy has become a widely used tool to investigate small calibre sensory nerves including somatic unmyelinated intraepidermal nerve fibres (IENF), dermal myelinated nerve fibres, and autonomic nerve fibres in peripheral neuropathies and other conditions. Different techniques for tissue processing and nerve fibre evaluation have been used. In March 2004, a Task Force was set up under the auspices of the European Federation of Neurological Societies (EFNS) with the aim of developing guidelines on the use of skin biopsy in the diagnosis of peripheral neuropathies. We searched the Medline database from 1989, the year of the first publication describing the innervation of human skin using immunostaining with anti-protein-gene-product 9.5 (PGP 9.5) antibodies, to 31 March 2005. All pertinent papers were rated according to the EFNS guidance. The final version of the guidelines was elaborated after consensus amongst members of the Task Force was reached. For diagnostic purposes in peripheral neuropathies, we recommend performing a 3-mm punch skin biopsy at the distal leg and quantifying the linear density of IENF in at least three 50-mum thick sections per biopsy, fixed in 2% PLP or Zamboni's solution, by bright-field immunohistochemistry or immunofluorescence with anti-PGP 9.5 antibodies (level A recommendation). Quantification of IENF density closely correlated with warm and heat-pain threshold, and appeared more sensitive than sensory nerve conduction study and sural nerve biopsy in diagnosing small-fibre sensory neuropathy. Diagnostic efficiency and predictive values of this technique were very high (level A recommendation). Confocal microscopy may be particularly useful to investigate myelinated nerve fibres, dermal receptors and dermal annex innervation. In future, the diagnostic yield of dermal myelinated nerve fibre quantification and of sweat gland innervation should be addressed. Longitudinal studies of IENF density and regeneration rate are warranted to correlate neuropathological changes with progression of neuropathy and to assess the potential usefulness of skin biopsy as an outcome measure in peripheral neuropathy trials (level B recommendation). In conclusion, punch skin biopsy is a safe and reliable technique (level A recommendation). Training in an established cutaneous nerve laboratory is recommended before using skin biopsy as a diagnostic tool in peripheral neuropathies. Quality control at all levels is mandatory.

Imunofluorescência para neuropeptídeos na mucosa nasal humana: avaliação de técnica para peptídeo intestinal vasoativo (VIP)

Os neuropeptídeos são neurotransmissores relevantes na fisiologia nasal e o conhecimento crescente acerca de seu papel na fisiopatologia de doenças nasais abre novas perspectivas. A sua investigação na mucosa nasal humana baseia-se em grande parte em marcação imunológica, método complexo e sujeito a inúmeros fatores de erro. Com o intuito de viabilizar este tipo de pesquisa em nosso meio, um método de imunofluorescência para peptídeo intestinal vasoativo (VIP) na mucosa nasal humana é proposto e avaliado. FORMA DE ESTUDO: Coorte transversal. MATERIAL E MÉTODO: Oito pacientes submetidos a cirurgia funcional do nariz têm um fragmento de mucosa coletado da concha inferior. O tecido foi fixado em solução de Zamboni (paraformaldeído 4% tamponado e ácido pícrico 0,4%), congelado em nitrogênio líquido e armazenado. Cortes de 14 µm foram realizados e submetidos à reação de imunofluorescência para VIP (Península Laboratories). As imagens microscópicas foram documentadas em fotografia convencional. A especificidade, sensibilidade e reprodutibilidade de execução foram avaliadas. A reprodutibilidade de interpretação de resultados foi avaliada através da comparação de graus de marcação (0 a 4) atribuídos às fotos por seis observadores. RESULTADOS: Os resultados mostraram ser o método suficientemente específico, sensível, além de reprodutível em sua execução. A interpretação de resultados mostrou depender do perfeito esclarecimento do observador no julgamento das imagens de imunofluorescência, mas mostrou uniformidade. CONCLUSÃO: O método proposto foi considerado útil na pesquisa de neuropeptídeos na mucosa nasal humana.

Neuropeptide immunofluorescence in human nasal mucosa: assessment of the technique for vasoactive intestinal peptide (VIP)

SummaryNeuropeptides are important neurotransmitters in nasal physiology and the increasing knowledge of their role in nasal diseases brings new therapeutic perspectives. The investigation of human nasal mucosa neuropeptides is based mostly on immunocytochemistry, a complex approach whose resulting factors may be variable. Aiming to make this kind of research available, an immunofluorescence approach for vasoactive intestinal peptide (VIP) in human nasal mucosa is proposed and evaluated.Study designTransversal cohort.Material and MethodHuman inferior turbinate samples were obtained at time of nasal surgery from eight patients. The samples were fixed in Zamboni solution (4% phosphate-buffered paraformaldehyde and 0.4% picric acid), snap-frozen and stored at -70ºC. 14 µm sections were then obtained. Immunofluorescence staining for VIP (Peninsula Laboratories) was performed and its images documented by conventional photography. The method's specificity, sensitivity and reproducibility of execution were evaluated. Additionally, the reproducibility of interpretation of results was evaluated through the comparison of staining scores (0 to 4) attributed to the images by six observers.ResultsThe results showed the approach to be very specific and sensible, besides being reproducible in its execution. The interpretation of results may depend on the observer's accuracy in judging immunofluorescence images, but it showed uniformity.ConclusionThe proposed method was highly useful for research purposes in neuropeptides in human nasal mucosa.

Hypoxia correlates with angiogenesis in cervical cancers

Tissue hypoxia stimulates the induction of the angiogenic substances vascular endothelial growth factor and erythropoietin in the locus of the tissue. We have previously demonstrated that erythropoietin promotes angiogenesis by binding to its receptor in the endothelial cells of uterine and ovarian malignancies. In the present study, we examined whether malignant uterine cervix tissue showed hypoxia and whether hypoxia correlated with high vascular density through vascular endothelial growth factor. To detect tissue hypoxia, we estimated the content of ATP in squamous cell carcinoma of the uterine cervix and in the normal cervix, using liquid chromatography columns. Surgically resected samples were fixed in Zamboni solution and processed for immunohistochemical microscopy to identify the endothelial cells and the location of vascular endothelial growth factor, with the use of anti-factor VIII and anti-vascular endothelial growth factor165 antibody, respectively. The microvessels in a definite area were counted in sections of each specimen. Significantly lower ATP levels and significantly higher vascular density were seen in squamous cell carcinoma than in the controls (P<0.05). The microvessel number in relation to ATP content was significantly higher in squamous cell carcinoma than in the controls (P<0.001). Moreover vascular endothelial growth factor, the hyperplastic epithelium of the squamous cell carcinoma contained the immunoreactivity, with characteristic histopathological features suggesting retention of tissue fluid. Squamous cell carcinoma of the uterine cervix showed hypoxia which correlated with abundant vascularity. Vascular endothelial growth factor expressed in the hyperplastic epithelium appears to promote angiogenesis in squamous cell carcinoma of the uterine cervix.

Autonomic nerve changes in the mouse pancreas after pancreatic duct ligation.

The mouse pancreas exhibits distinct atrophy of the exocrine tissue following pancreatic duct ligation. To investigate changes of innervation in the whole pancreas after pancreatic duct ligation. The mouse pancreatic duct was ligated at 6 weeks of age. Pancreatic tissues were removed 7 days and 14 days after the ligation, fixed by perfusion and immersion with Zamboni solution, and embedded in gelatin. The whole organ was serially sectioned at a thickness of 100 microm, histochemically stained for cholinesterase, and observed by light microscopy. The number and volume of intrapancreatic ganglia, number of ganglion cells, and volume of each ganglion cell in the whole pancreas were quantitated. Some sections were analyzed using transmission electron microscopy after histochemically staining for cholinesterase. In the normal pancreas, ganglia were often situated on the outer surface of the islets of Langerhans. Thick nerve bundles ran along the arteries and emanated thin nerve fibers that surrounded the arterioles. In the atrophied pancreas following pancreatic duct ligation, ganglia remained on the islets of Langerhans as in normal mice, while the nerve fibers appeared dense, bending and curling in a more complex manner. The thin nerves also crossed each other in a complex network. Using morphometry in the pancreas following pancreatic duct ligation, the total ganglion cell number was found to decrease from normal levels. The mean ganglion cell volume in the ligated pancreas was significantly smaller than that in normal mice. As observed by transmission electron microscopy, some ganglion cells in the ligated pancreas were negative for cholinesterase activity but were surrounded by positive staining around the surface. These results suggest that the function of pancreatic ganglion cells changes with organ atrophy after pancreatic duct ligation.

Immersion fixation with Carnoy solution for conventional immunohistochemical detection of particular N-methyl-D-aspartate receptor subunits in murine hippocampus.

Immunoblotting analysis revealed heterologous distribution profiles of the N-methyl-D-aspartate (NMDA) receptor subunits GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 in membrane preparations of different murine brain structures, with exclusive detection of GluR epsilon-3 subunit in cerebellar preparations. Mice were anesthetized and perfused with 4% paraformaldehyde (PA), Zamboni, or Carnoy solution for subsequent immunohistochemical detection of these NMDA receptor subunits. In coronal brain sections from animals perfused with 4% PA and Zamboni solutions, high immunoreactivity was detected exclusively with GluR zeta-1, GluR epsilon-1, GluR epsilon-2, and GluR epsilon-3 subunits in the pyramidal and dentate granular layers, where the GluR epsilon-3 subunit is supposed to be absent. By contrast, high immunoreactivity was detected with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2, but not GluR epsilon-3, subunits in the strata oriens and radiatum of the CA1 subfield without immunoreactivity along the pyramidal and granular layers when sections were prepared by immersion fixation with Carnoy solution after dissection from brains of mice decapitated. On these sections, relatively high immunoreactivity was found also with GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the stratum lacunosum-moleculare of the CA1 region, the strata oriens, radiatum, and lacunosum-moleculare of the CA3 region, and the stratum moleculare of the dentate gyrus, respectively. The systemic administration of kainate led to significant decreases in immunoreactivity to GluR zeta-1, GluR epsilon-1, and GluR epsilon-2 subunits in the CA1 and CA3 subfields on brain sections prepared by immersion fixation with Carnoy solution. These results suggest that immersion fixation with Carnoy solution may be suitable and appropriate for reproducible and quantitative immunohistochemical detection of particular NMDA receptor subunits in murine hippocampus.

Effect of Chagas' disease on nitric oxide-containing neurons in severely affected and unaffected intestine.

The pathophysiology of Chagas' disease is incompletely understood. Neuronal nitric oxide has been cited as a candidate neurotransmitter responsible for relaxation of the internal anal sphincter. Neuronal nicotinamide adenine dinucleotide phosphate diaphorase can be used as a marker for neuronal nitric oxide synthase. This study was designed to examine the alterations of the nitric oxide-containing neurons in the enteric nervous system of the colon of patients who underwent resections for advanced megacolon and to compare these specimens with small-bowel specimens from the same patients and with specimens from control subjects. Specimens from resected rectum and extramucosal small-bowel biopsy specimens from 11 patients with Chagas megacolon but no apparent small-bowel clinical involvement were compared with the uninvolved colon and jejunum of 10 control patients with colon cancer. Tissues were fixed in Zamboni solution and evaluated by histochemistry for nicotinamide adenine dinucleotide phosphate diaphorase-containing neurons. Reactivity was evaluated on a 0 to 4 scale in the longitudinal muscle, myenteric plexus, circular muscle, submucosal plexus, and mucosa. Specimens from control patients showed well-stained myenteric and submucosal neurons and an abundant network of terminal nerve fibers in the muscle layers. Chagasic specimens had decreased staining in all layers of the gut. Overall there was a statistically significant decrease in nicotinamide adenine dinucleotide phosphate diaphorase-containing neurons. Biopsy specimens from clinically uninvolved small bowel of patients with Chagas' disease also showed decreased reactivity, but to a lesser degree. Nicotinamide adenine dinucleotide phosphate diaphorase activity is decreased in patients with advanced megacolon. The alterations are more relevant in the myenteric plexus and the circular muscle. Reactivity is also diminished in the clinically uninvolved small bowel, but to a lesser extent.

Preparation of Heat-Labile Antigens on Air-Dried Cytologic Specimens for Immunocytochemical Analysis

To demonstrate the efficacy of Zamboni's solution and storage medium for immunocytochemistry of Ki-67, p53 tumor suppressor gene product and CD3 on long-preserved, air-dried cytologic specimens. Air-dried imprint cytologic specimens obtained from 13 lymphatic tissues and 13 colorectal cancers were fixed with Zamboni's solution and stored for various periods in storage medium including 50% glycerol at -10 to -20 degree C. Mouse monoclonal antibody PC, PAb 1801 and anti-Leu-4 were used for immunostaining. Immunocytochemical identification of Ki-67, p53 and CD3 was possible on specimens stored for 36, 32 and 16 weeks, respectively. Our method, using Zamboni's solution and storage medium, is feasible for immunocytochemistry on long-preserved, air-dried cytologic specimens using the three antibodies were employed.

Immunohistochemical differentiation of monoclonal GAD antibodies recognizing linear or conformational epitope regions.

GAD65 is targeted by different patterns of autoantibodies [glutamic acid decarboxylase (GAD)-AAbs] in insulin-dependent diabetes mellitus (IDDM) and stiff-man syndrome (SMS). To study differentiation of the GAD-AAb pattern by immunohistochemistry, we examined the immunostaining of 15 monoclonal GAD antibodies (mc-GAD-Abs), which recognized different epitope regions of the antigen, on human pancreatic sections that were unfixed or fixed with different fixatives. By a competitive sandwich enzyme-linked immunosorbent assay (ELISA), three binding patterns of mc-GAD-Abs were identified: 5 of 15 mc-GAD-Abs recognized a linear N-terminal epitope (p1), 5 of 15 were reactive with a conformational GAD65 epitope region (p2), and 5 of 15 were cross-reactive with GAD67 (p3). These patterns of mc-GAD-Abs were tested for islet cell binding by indirect immunofluorescence on pancreatic sections treated with either (1) Bouin's solution, (2) Zamboni's solution, or (3) phosphate-buffered formaldehyde for 0.5, 1, 2, and 18 h at 4 degrees C. After fixation for up to 2 h no differentiation of immunoreactivity of patterns was observed using the three fixatives. mc-GAD-Abs recognizing conformational epitope regions (p2) revealed a marked reduced immunoreactivity on pancreatic sections fixed for 18 h with 4% formaldehyde, while mc-GAD-Abs reactive with linear epitopes (p1, p3) were detectable with strong binding. This fixation procedure was used to compare the immunoreactivity of GAD-AAb+ or GAD-AAb- islet cell cytoplasmic antibody-positive (ICA+) sera of IDDM (n = 27) and SMS patients (n = 3). The three SMS sera were reactive with GAD on fixed islets but showed a reduced titer, whereas the majority of IDDM sera (22/27; 81.5%) were not detectable; 70.6% (12/ 17) of GAD-AAb+ IDDM sera were not detectable on fixed islets. Furthermore, all 10 GAD-AAb- IDDM sera tested failed to react with fixed pancreas, which also suggested an alteration of non-GAD-ICA antigens. In conclusion, the fixation of human pancreatic sections with formaldehyde for 18 h allows the differentiation of GAD-AAbs recognizing linear and conformational epitope regions.

Trichinella spiralis: Ultrastructural localization of a target antigen recognized by a monoclonal antibody

The localization of a 160-kDa molecule recognized by a monoclonal antibody of the IgG 1-class (TS32D12) to Trichinella spirilis was demonstrated at the subcellular level. Mature muscle larvae recovered from infected mice were fixed with Zamboni solution, and embedded in LR white resin. Ultrathin sections were incubated with TS32D12 and subsequently stained using a secondary antibody-coated colloidal gold probe. TS32D12 reacted to constituents in the α-stichosome cells of the parasite; that is, α-granules and rough endoplasmic reticulum. Other structures were not recognized by the immunogold probe, but occasionally weak positive staining was seen on β-granules, the cuticle, hypodermis and myofibrils.

In situ fixation of the neonatal brain and spinal cord.

A delay in the autopsy can result in significant tissue autolysis, especially in the central nervous system. We have developed a rapid technique of in situ fixation that preserves central nervous system tissues until the formal autopsy can be performed. Through the lateral margin of the anterior fontanelle, Zamboni's solution is injected percutaneously into the lateral ventricles and allowed to exit via an intrathecal spinal needle. The choice of fixative allows a wide array of postmortem studies to be done.

Subcellular localization of the antigenic sites of relaxin in the luteal cells of the pregnant rat using an improved immunocytochemical technique

Using an improved immunocytochemical technique, the present work has demonstrated the antigenic sites of relaxin in the luteal cells of the pregnant rat with preservation of the ultrastructural details. Tissues collected were fixed with either 4% paraformaldehyde (PFA), 4% PFA containing 0.3% glutaraldehyde (GLA) or Zamboni solution followed or not by 1% osmium. Ultrathin sections of the tissues embedded in epoxy resin were incubated with rat relaxin antiserum and antibody binding sites were visualized with protein A-gold. Among these fixatives, the use of 4% PFA-0.3% GLA, followed by osmium, allowed excellent preservation of fine cellular structure with maintenance of antigenic activity and immobilization of the antigen. The majority of the gold particles indicative of relaxin immunoreactivity were localized on the dense core of the membrane-limited granules, while a minor fraction was localized on the Golgi apparatus. The results suggest that the subcellular localization of relaxin can be detected after post-fixation with osmium and that the antigenic sites of relaxin exist mostly in the dense granules with a small fraction in the Golgi apparatus.

Immunolocalization of mineralocorticoid receptor in human kidney, pancreas, salivary, mammary and sweat glands: a light and electron microscopic immunohistochemical study.

Using a polyclonal antibody against a synthetic fusion protein corresponding to 167 amino acids of the N-terminal region of the human renal mineralocorticoid receptor (MR), an immunohistochemical study was performed to investigate the intraglandular and intracellular localization of the receptor in human kidney, salivary gland, pancreas, mammary gland and sweat gland both at the light and electron microscopic levels. In the kidney, immunoreactivity was observed in distal convoluted tubules, branches of Henle's loop, and collecting tubules in the renal cortex, and papillary and Henle's loops' ducts in the renal medulla. No significant differences in the distribution of immunoreactivity were observed using different fixatives (10% neutral formalin, 100% methanol, 4% paraformaldehyde, PLP (periodate-lysine-2% paraformaldehyde) solution and Zamboni solution) and processing methods (paraffin embedding and frozen sectioning). Immunoreactivity in the kidney was observed in both the cytoplasm and nucleus, with cytoplasmic staining predominant, regardless of the methods of tissue preparation. Immunoelectron microscopy, employing a pre-embedding method, demonstrated the presence of immunoreaction precipitates in nuclei, endoplasmic reticulum, perinuclear cisternae, free cytoplasm and cell membranes. In nuclei, immunoreactivity was observed in euchromatin but not in heterochromatin, which is consistent with an association of MR with specific DNA regulatory elements located in transcriptionally active euchromatin. In other organs, MR was expressed in cells of the excretory ductal system where mineralocorticoids are known to play a role in electrolyte homeostasis.

&lt;b&gt;CORRECTION:&lt;/b&gt; Calcitonin gene-related peptide-immunoreactive nerves in carcinoma experimentally induced in the hamster tongue, Biomedical Research 12 (6) 377-390

The localization and distribution of Met-enhephalin (M-ENK)-, Leu-enhephalin (L-ENK)- and Met-enkephalin-Arg-Gly-Leu (M-ENK-AGL)-like immunoreactivities (LI) in the guinea pig pancreas were studied by means of Sternberger's peroxidase-antiperoxidase (PAP) method. Hartley Strain guinea pigs were used in this study. The pancreas was perfusion-fixed with Zamboni's solution -and tissues were processed for immunostainings. Adrenal medullary cells were employed as staining control. Immunoreactivitres for M-ENK, L-EMK and M-ENK-AGL, proenhephalin A-derived opioid peptides, were localised in nerve elements with a similar distribution pattern