Simple Immunohistochemical Method Research Articles

Types of Neurons in the Human Colonic Myenteric Plexus Identified by Multilayer Immunohistochemical Coding

Gut functions including motility, secretion, and blood flow are largely controlled by the enteric nervous system. Characterizing the different classes of enteric neurons in the human gut is an important step to understand how its circuitry is organized and how it is affected by disease. Using multiplexed immunohistochemistry, 12 discriminating antisera were applied to distinguish different classes of myenteric neurons in the human colon (2596 neurons, 12 patients) according to their chemical coding. All antisera were applied to every neuron, in multiple layers, separated by elutions. A total of 164 combinations of immunohistochemical markers were present among the 2596 neurons, which could be divided into 20 classes, with statistical validation. Putative functions were ascribed for 4 classes of putative excitatory motor neurons (EMN1-4), 4 inhibitory motor neurons (IMN1-4), 3 ascending interneurons (AIN1-3), 6 descending interneurons (DIN1-6), 2 classes of multiaxonal sensory neurons (SN1-2), and a small, miscellaneous group (1.8% of total). Soma-dendritic morphology was analyzed, revealing 5 common shapes distributed differentially between the 20 classes. Distinctive baskets of axonal varicosities surrounded 45% of myenteric nerve cell bodies and were associated with close appositions, suggesting possible connectivity. Baskets of cholinergic terminals and several other types of baskets selectively targeted ascending interneurons and excitatory motor neurons but were significantly sparser around inhibitory motor neurons. Using a simple immunohistochemical method, human myenteric neurons were shown to comprise multiple classes based on chemical coding and morphology and dense clusters of axonal varicosities were selectively associated with some classes.

A simple immunohistochemical method for detecting microglia in rat brain

A simple immunohistochemical method for detecting microglia in rat brain

EPro-ClearSee: a simple immunohistochemical method that does not require sectioning of plant samples

Investigations into the epigenetic status of individual cells within tissues can produce both epigenetic data for different cell types and positional information of the cells. Thus, these investigations are important for understanding the intra- and inter-cellular control systems of developmental and environmental responses in plants. However, a simple method to detect epigenetic modifications of individual cells in plant tissues is not yet available because detection of the modifications requires immunohistochemistry using specific antibodies. In this study, we developed a simple immunohistochemical method that does not require sectioning to investigate epigenetic modifications. This method uses a clearing system to detect methylated histones, acetylated histones, methylated DNA and/or centromeric histone H3 variants. Analyses of four dicots and five monocots indicated that this method provides a universal technique to investigate epigenetic modifications in diverse plant species.

Association of celiac disease and intestinal lymphomas and other cancers

Celiac disease (CD) is associated with intestinal lymphoma and other forms of cancer, especially adenocarcinoma of the small intestine, of the pharynx, and of the esophagus. Enteropathy-associated T-cell lymphoma (EATL) is a rare form of high-grade, T-cell non-Hodgkin lymphoma (NHL) of the upper small intestine that is specifically associated with CD. This NHL subtype arises in patients with either previously or concomitantly diagnosed CD. In a subgroup of patients, there is progressive deterioration of a refractory form of CD. EATL derives from a clonal proliferation of intraepithelial lymphocytes and is often disseminated at diagnosis. Extraintestinal presentations are not uncommon in the liver/spleen, thyroid, skin, nasal sinus, and brain. The outlook of EATL is poor. Recent studies indicated that (1) CD is associated with a significantly increased risk for NHL, especially of the T-cell type and primarily localized in the gut (EATL); (2) the CD-lymphoma association is less common than previously thought, with a relative risk close to 3; (3) CD screening is not required in patients with NHL of any primary site at the onset, unless suggested by specific findings (T-cell origin and/or primary gut localization). The risk of NHL associated with clinically milder (or silent) forms could be lower than in typical cases of CD. Several follow-up studies suggest that the GFD protects from cancer development, especially if started during the first years of life. Strict adherence to the GFD seems to be the only possibility of preventing a subset of rare but very aggressive forms of cancer.

A novel immunohistochemical method for evaluation of antibody specificity and detection of labile targets in biological tissue

Immunohistochemistry is a key tool for analyzing target molecule localization within tissues. However, accurate results require an antibody that can distinguish between similar compounds. We present a simple immunohistochemical method that can also be used to rapidly evaluate antibodies' specificities. We demonstrate this technique with serotonin, an extremely labile compound. Serotonin (5-hydroxytryptamine, 5HT) is an important neurotransmitter regulating normal cognition and several mental disorders, as well as tumor growth, cardiopathology, and embryogenesis. Immunohistochemical detection of serotonin is commonly used as a neuronal cell marker and to provide crucial information on serotonin's role as an embryonic morphogen. It is necessary to be able to distinguish serotonin from closely related molecules with significantly different biological activity. Using our method, we identify antibodies that are specific for serotonin and show that some commercial 5HT antibodies often used to identify serotonergic cells in published papers are not 5HT-specific. These data demonstrate the necessity of specifically testing antibodies (especially in areas of high clinical relevance such as 5HT). We also illustrate detection of serotonergic cells in embryonic tissue using our technique. This method offers a number of general advantages for testing specificity of antibodies to any biological molecule, and helps avoid false positives and negatives during immunohistochemistry.

Distinction between coeliac disease and refractory sprue: a simple immunohistochemical method.

We recently showed that refractory sprue is distinct from coeliac disease, the former being characterized by abnormal intraepithelial T-lymphocytes expressing a cytoplasmic CD3 chain (CD3c), lacking CD3 and CD8 surface expression, and showing TCRgamma gene rearrangements. To take advantage of the abnormal phenotype of CD3c + CD8 - intraepithelial lymphocytes (IEL) in refractory sprue we developed a simple method to distinguish coeliac disease from refractory sprue. Comparative immunohistochemical studies using anti-CD3 and anti-CD8 antibodies were applied on paraffin-embedded and frozen biopsy specimens in refractory sprue (n = 6), coeliac disease (n = 10), healthy controls (n = 5) and suspected refractory sprue (n = 6). Comparable results were obtained on fixed and frozen biopsy specimens. In four of the six patients with suspected refractory sprue, abnormal CD3c + CD8 - IEL and TCRgamma gene rearrangements were found, as in refractory sprue; the remaining two patients had normal (CD3 + CD8 +) IEL and no TCRgamma gene rearrangements. Both patients had coeliac disease, as one failed to comply with a gluten-free diet, while the other was a slow responder. This simplified immunostaining method using anti-CD3 and anti-CD8 antibodies on paraffin sections can distinguish active coeliac disease from refractory sprue and should prove useful in clinical practice.

Nuclear localization and mutation of beta-catenin in medulloblastomas.

The adenomatous polyposis coli (APC) gene, a member of the Wingless/Wnt signal transduction pathway, has been implicated in the development of medulloblastomas in Turcot's syndrome. beta-catenin also functions in this highly conserved signaling pathway and is instrumental in growth and development. Mutations in either APC or beta-catenin can stabilize beta-catenin protein. Stabilized beta-catenin complexes with Tcf/Lef transcription factors and moves from the cytoplasm into the nucleus where it regulates the transcription of c-Myc and other genes. Nuclear localization of beta-catenin therefore implies activation of the signaling pathway. We have analyzed the subcellular localization of beta-catenin in 51 sporadic medulloblastomas and in 1 medulloblastoma arising in a patient with Turcot's syndrome. Nuclear beta-catenin staining was present in 9 of the sporadic tumors (18%) and in the 1 medulloblastoma from a Turcot's patient. The remaining 41 cases did not show nuclear staining. This confirms earlier observations that Wingless/Wnt signaling is involved in a subset of sporadic medulloblastomas. We also examined 48 glial and meningeal CNS tumors, all of which were negative for nuclear beta-catenin. Exon 3 of beta-catenin was sequenced in 6 of the 9 sporadic medulloblastomas with nuclear beta-catenin staining. Five of the 6 tumors sequenced had mutations affecting highly conserved beta-catenin phosphorylation sites involved in protein stability. These data suggest a simple immunohistochemical method to screen for beta-catenin mutations in medulloblastomas.

PCNA and Ki-67 labelling indices in pre-irradiated and post-irradiated astrocytomas: a comparative immunohistochemical analysis for evaluation of proliferative activity.

AIM: To determine the tumour proliferative activity in a series of archival cerebral astrocytomas and compare proliferating cell nuclear antigen (PCNA) and Ki-67 labelling indices in the primary and recurring...

A new multilabeling technique for simultaneous demonstration of different islet cells in permanent slides.

We describe a simple immunohistochemical method for simultaneous demonstration of insulin, glucagon, somatostatin, and pancreatic polypeptide cells of the Langerhans islet in routinely fixed paraffin-embedded human and Syrian hamster pancreases. With the use of alcohol insoluble chromogens, the slides can be mounted in Permount and be kept permanently without losing the color quality and intensity. With this technique, the spatial topographical distribution and morphology of the individual pancreatic endocrine cells can be investigated in the healthy and diseased pancreas.

Cell kinetics in skin disorders with disturbed keratinization.

A relatively simple immunohistochemical method was developed and used on cryostat sections. The monoclonal antibody Ki67 was used as marker for actively cycling cells and Pab601 for germinative cells. Counts were expressed as Ki67- or Pab601-positive cells/mm. In order to improve our understanding of the pathogenetic mechanisms in skin disorders with disturbed keratinization we have measured cell kinetic values in dyskeratosis follicularis, pemphigus benigna familiaris chronica, autosomal dominant ichthyosis vulgaris, X-linked recessive ichthyosis, atopic dermatitis and psoriasis and compared them with previous values derived with autoradiography using tritiated thymidine. The results showed that microscopical acanthosis is related to an increase of the germinative population, while the increased epidermal turnover is associated with increased numbers of cycling cells. The cell kinetic changes seem to be all secondary except in psoriasis where a dysregulation in the epidermal growth may cause the epidermal changes. This simple method allows quick evaluation of drug efficacy which might be useful in atopic dermatitis and psoriasis.