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Immunohistochemistry (IHC) Application Guide       免疫组化(IHC)应用指南 免疫组化(IHC),是应用免疫学基本原理——抗原抗体反应,即抗原与抗体特异性结合的原理,通过化学反应使标记抗体的显色剂(荧光素、酶、金属离子、同位素)显色来确定组织细胞内抗原(多肽和蛋白质),对其进行定位、定性及定量的研究,称为免疫组织化学技术(immunohistochemistry)或免疫细胞化学技术(immunocytochemistry)。 Easily plan and optimize your IHC experiments. The booklet includes tips and guidelines on: Sample...
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2015年01月09日 免疫技术 ⁄ 被围观 1,791+
Fixation immobilizes antigens while retaining cellular and subcellular structure. The choice of fixation method used depends on the sensitivity of the epitope as well as the antibodies themselves and may require some optimization. Print this protocol. IHC fixation can be done using crosslinking reagents, such as paraformaldehyde. These are better at preserving cell structure, but may reduce the antigenicity of some cell components as the crosslinking can obstruct antibod...
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2015年01月02日 免疫技术 ⁄ 被围观 1,855+
Once the tissue is fixed, it needs to be processed so that it is adequately supported for cutting into sections of up to 5 µm thickness. The tissue is dehydrated, cleared and then infiltrated with medium to enable sectioning. Paraffin wax is the most common medium used for immunostaining. Print this protocol. Paraffin tissue processing After fixation, rinse tissue with PBS until fixative is completely removed. Dehydrate tissue using ethanol in the following sequence....
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2014年12月31日 免疫技术 ⁄ 被围观 1,510+
Deparaffinization is only required when using paraffin embedded sections. Before proceeding with the staining protocol, the slides must be deparaffinized and rehydrated. Incomplete removal of paraffin can lead to poor staining of the section. Print this protocol. ​ Materials and reagents Xylene 100% ethanol 95% ethanol ​70% ethanol 50% ethanol Method Place the slides in a rack, and perform the following washes using Coplins jar: Solution Incubation time Xylene ...
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2014年12月30日 免疫技术 ⁄ 被围观 1,589+
  Permeabilization is only required when the antibody needs access to the inside of the cells to detect the protein. Cytoskeletal, viral and some enzyme antigens usually give optimal results when fixed with acetone, ethanol or formaldehyde (high concentration). Print this protocol. Antigens in cytoplasmic organelles and granules will require a fixation and permeabilization method depending on the antigen. Please refer to the antibody datasheet for recommendations. ...
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2014年12月29日 免疫技术 ⁄ 被围观 1,938+
Most formalin-fixed tissues require an antigen retrieval step before immunohistochemical staining can proceed. This is due to the formation of methylene bridges during fixation, which cross-link proteins and therefore mask antigenic sites. The two methods of antigen retrieval are heat-mediated (also known as heat-induced epitope retrieval or HIER) and enzymatic. Print this protocol. Both HIER and enzymatic antigen retrieval serve to break the methylene bridges and expos...
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Print this protocol. Paraffin and frozen sections Reagents can be applied manually by pipette or the sequence of the protocol can be adapted to automated and semi-automated systems if these are available. All incubations should be carried out in a humidified chamber to avoid drying of the tissue. Drying at any stage will lead to non-specific binding and ultimately high background staining. A shallow, plastic box with a sealed lid and wet tissue paper in the bottom is ...
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2014年12月25日 免疫技术 ⁄ 被围观 1,706+
  Protocol and troubleshooting tips for whole mount immunohistochemical staining. Whole mount staining is the staining of small pieces of tissue, usually embryos, without sectioning onto slides first. This is often used on embryos by stem cell and embryonic development researchers and also neuroscientists who are able to stain the whole embryos at various stages to follow the expression of target proteins through the development of the animal. Whole mount staining ...
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Guideline procedure for staining of cell cultures using immunofluoresence. 电子版下载: Print this protocol. General procedure Coat coverslips with polyethyleneimine or poly-L-lysine for 1 hr at room temperature. Rinse coverslips well with sterile H2O (three times 5 min each). Allow coverslips to dry completely and sterilize them under UV light for at least 4 hr. Grow cells on glass coverslips or prepare cytospin or smear preparation. Rinse briefly in phosphate-buf...
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2014年12月20日 免疫技术 ⁄ 被围观 1,453+
Tips for eliminating background when staining mouse tissue with a mouse monoclonal antibody. 电子版下载:Print this protocol. Staining of mouse tissue using mouse antibody is a complicated process as high levels of background are often observed. It is notoriously difficult to eliminate this background. Much of the background is caused by secondary antibody binding to endogenous mouse IgG in the tissue being stained, and to Fc receptors on B cells, plasma cells and macrop...
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