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In Situ Hybridization Protocols for the Brain, Volume 47
Contact Us. Tissue samples, tissue arrays, cells, microorganisms, probes and services for your research. Creative Bioarray's routine and custom ISH services are flexible to meet your needs as follows: Preparation of Tissue Sections We accept shipment of formalin-fixed tissues, cell samples or unstained slides. Following embedment would be performed. Frozen Embedding and Sectioning Paraffin Embedding and Sectioning Cell Pellet Embedding and Sectioning Probe Design, Labeling and Purification Synthesis of DNA and RNA probes isotopic and non-isotopic Hybridization Optimization of hybridization conditions; Hybridization of sense and anti-sense probes with the tissue of interest ISH Staining Using proprietary reagents and methods Reporting Final report with digital images and stained slides Quality Control Due to the inherent complexity of this technique, appropriate labeling and hybridization controls are always included in each project.
In particular, control probes of housekeeping genes are applied to determine the mRNA quality and abundance in tissues provided by customers. Frequently, we use control tissues to validate the results. Consultation Comprehensive pathology consultation from our experienced staff pathologists is also available upon request. Related Sections Services: Biosample Services. Cell-Based Drug Discovery.
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Bodkin, D. Methods 10 , 45— Giros, B. Nature , — Lewis, M. Synapse 2 , — Young III, W. A Practical Approach Wilkinson, D. Biological Techniques. Academic Press, San Diego. Sernia, C.
Conway, S. Molecular Diagnosis of Cancer. Cotter, F. Sharif, N. IRL, Oxford. Lewis, E.
Mantmayeur, J. Comparison between autoclaving and PK treatment for antigen retrieval. Representative sections that underwent antigen retrieval by A autoclaving and B PK treatment. C Mean IOD ratio of each group. The Malat1-b probe was used to detect the expression of malat1 Fig. Using immuno-FISH, malat1 and three different proteins were successfully co-detected. NeuN-positive cells were double labeled with malat1 green-red merge; Fig. These results indicated that Malat1 is expressed in neurons but not in microglia or astrocytes.
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Double labeling for Malat1 and protein markers. Only NeuN-positive cells were double labeled. It is typically used to detect the localization and abundance of target RNA expression at the histological level 9. Many lncRNAs have been implicated in the functioning of the nervous system and development of disease In the present study, a modified protocol of immuno-FISH was developed. Using this protocol, high-quality fluorescent signals and histological data were obtained that provided information about the expression and distribution of lncRNA malat1 and three different proteins in the spinal cord.
Pretreatment of tissue sections is a critical step for obtaining satisfactory fluorescence signals, and this step is modified in different protocols. The purpose of pretreatment is to ensure signal specificity and minimize non-specific background signals 14 , Acetylation, which negatively charges sections to reduce the adsorption of negatively-charged probes, is widely used as a pretreatment step to reduce background staining 10 , 16 , However, in some protocols, acetylation has been reported as a dispensable step 10 , As such, in the present study, sections were not treated with acetylation, which simplified the protocol and saved time.
However, previous results have indicated that PK may also denature proteins of interest, which makes it incompatible with immunofluorescence 9. In the present study, sodium citrate-based antigen unmasking and autoclaving were used for antigen retrieval, and high-quality FISH and immunofluorescence signals were obtained.
High temperatures may also denature membrane proteins and RNA-protein complexes, unmasking the antigenic sites of target proteins and thus improving antibody-antigen reactions In addition, high pressure prevents solutions from boiling, which otherwise causes tissue sections to peel away from slides. The reagents used in the current protocol were readily available, relatively non-toxic and inexpensive. Researchers may prepare the antigen unmasking, prehybridization, and hybridization buffers using common reagents following the simple formulas listed above.
Another advantage of the current protocol was that it was time-efficient. All of the steps undertaken in the present study were performed within 24—32 h. The variation in the time required to complete the protocol was mainly due to the antibodies used for immunofluorescence, as different antigen-antibody reactions required different incubation periods.
Malat1 is an abundant lncRNA that has been demonstrated to regulate the progression of many diseases 4. In the present study, the distribution of malat1 in the spinal cord of rats was assessed using a simple immuno-FISH protocol, and it was demonstrated that malat1 was expressed in neurons but not in microglia or astrocytes.
Despite the lack of a signal amplification method, satisfactory fluorescence signals were obtained. However, the abundance of some lncRNAs is low, for example BACE1-AS and lnc-DC 21 , 22 , and in these cases signal amplification treatments, including tyramide amplification and specially designed riboprobes, may be necessary to obtain detectable fluorescence. In conclusion, the present study performed a simple immuno-FISH protocol for the detection of malat1 lncRNA and protein markers of neurons, microglia and astrocytes in frozen spinal cord sections of rats.
Advantages of the modified immune-FISH protocol include the ready availability of reagents and general speed of the method. The present study was supported by the Ministry of Science and Technology of China Program; grant no.
In situ hybridization on brain tissue.
Nat Neurosci. J Mol Med Berl. Cell Death Dis. Brain Pathol.