Immunohistochemistry (IHC)Staining Steps
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The document outlines the steps for DAKO IHC staining which includes deparaffinization and rehydration of the sample, antigen retrieval, blocking of endogenous peroxidase, incubation with primary and secondary antibodies, development with DAB, counterstaining with hematoxylin, and coverslipping. The process takes approximately 1-2 hours and involves rinsing between steps in various buffers and solvents.
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This document provides information about immunohistochemistry techniques. It discusses antigen retrieval methods like heat-induced epitope retrieval and protease digestion to expose buried epitopes. It describes primary and secondary antibody incubation steps and the differences between monoclonal and polyclonal antibodies. Detection methods like direct immunofluorescence, indirect immunofluorescence, peroxidase anti-peroxidase, and avidin-biotin complex are outlined. Chromogen substrates and counterstaining are also summarized. Automated immunohistochemistry systems and the differences between IHC and hematoxylin and eosin staining are briefly covered.IHC Link-Label
IHC Link-LabelVictoria Miller
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This document provides an overview of the steps in BioGenex's immunohistochemistry (IHC) staining protocol using their Super Sensitive Link-Label kits. The 11 key steps are: 1) baking tissue, 2) dewaxing, 3) antigen retrieval, 4) peroxide block, 5) power block, 6) primary antibody incubation, 7) link-label detection, 8) DAB chromogen, 9) hematoxylin counterstain, 10) mounting, and 11) washing steps between each stage. The document also includes examples of cytokeratin 18 staining and composition information for different Link-Label kit sizes and components.IHC protocols.pptx
IHC protocols.pptxMANJULRAJPUT1
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The immunohistochemistry protocol involves sectioning tissue samples onto slides, deparaffinizing the slides using xylene and ethanol, performing antigen retrieval in a retrieval buffer in a decloaking chamber, blocking peroxidase activity and washing in TBS buffer, incubating with primary and secondary antibodies, staining with hematoxylin and DAB chromogen, dehydrating and mounting the slides for microscopic examination.Immunohistochemistry
ImmunohistochemistryJaandeepCheema
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Immunohistochemistry is a technique that uses antibodies to identify antigens in cells of a tissue sample. It relies on the principle of antibodies binding specifically to antigens in cells. The primary antibody binds to the antigen of interest, while the secondary antibody is conjugated to an enzyme or fluorescent label for visualization. A chromogen is used to produce a colored precipitate that can be seen under the microscope. Immunohistochemistry has many applications in pathology for tumor diagnosis and classification by identifying cellular markers of differentiation. It allows identification of cell lineages and tumor types through characteristic protein expression patterns revealed by specific antibody staining.Haematoxylin__Eosin.ppt
Haematoxylin__Eosin.pptDr Sherin Nedumpillil
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Haematoxylin and eosin (H&E) staining is the most widely used histological stain. It uses haematoxylin, which stains nuclei blue, and eosin, which stains cytoplasm and extracellular components varying shades of pink. There are different types of haematoxylin depending on the mordant used, including alum, iron, tungsten, and un-mordanted haematoxylins. Alum haematoxylins like Mayer's and Harris' are most commonly used in H&E staining. Iron haematoxylins yield a stronger nuclear stain and can demonstrate more structures. Understanding the different haematoxylins allows their more informative use in diagnostic pathology.Immunohistochemistry Antibody Validation Report for Anti-Cleaved-Caspase-1 (D...
Immunohistochemistry Antibody Validation Report for Anti-Cleaved-Caspase-1 (D...St John's Laboratory Ltd
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The document details an immunohistochemical analysis protocol for various human tissues (breast, liver, kidney, and stomach cancer) using cleaved-caspase-1 (d210) polyclonal antibody. The protocol includes steps for tissue processing, primary and secondary antibody incubation, staining, and visualization, providing specific conditions for each stage to ensure accurate results. Each tissue type follows a similar method, with adjustments for antibody dilution and incubation times based on the specific application.Immunohistochemistry Antibody Validation Report for Anti-Phospho-CaMKII硫/粒/隆 ...
Immunohistochemistry Antibody Validation Report for Anti-Phospho-CaMKII硫/粒/隆 ...St John's Laboratory Ltd
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Immunohistochemistry Antibody Validation Report for Anti-Cleaved-Caspase-1 (D...
Immunohistochemistry Antibody Validation Report for Anti-Cleaved-Caspase-1 (D...St John's Laboratory Ltd
油
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Immunohistochemistry (IHC)Staining Steps
- 1. DAKO IHC Staining Steps 1.Deparaffinization and Rehydration 2.Rinse with Buffer 3.Target Retrieval Cool 4. Rinse in Tap Water and DW 5.Block Endogenous Peroxidase 6.Rinse with Buffer 7.Primary Antibody 8.Rinse with Buffer 9.Envision 10.Rinse with Buffer 11.DAB 12.Rinse with DW 13.Hematoxylin 14.Rinse with TW 15.Coverslip with Aqueous Resin
- 2. 1.Deparaffinization and Rehydration 1. Xylene - 5 mins. 2. Xylene - 5 mins. 3. Absolute ethanol -3 mins. 4. Absolute ethanol - 3 mins 5. 95% ethanol - 3 mins. 6. 95% ethanol - 3 mins. 7. Distilled water (30 seconds to 1 min.)
- 3. 3.Target Retrieval Total: 15 18 mins. plus 20 mins. cooling time = 35-38 mins) 1. High - 5 mins. 2. Medium High/Low - 5 mins. 3. Medium - 5 mins. 4. Medium - 3 mins. 5. Cooling time - 20 mins. 700-800 watts microwave oven
- 4. 5.Block Endogenous Peroxidase Hydrogen Peroxide 3% in TBST H2O2 Hydrogen Peroxide blocking - 5 mins . (Wash buffer 1 min.)
- 5. Primary Antibody 7.Primary Antibody 20 minutes
- 6. 8.Rinse with Buffer Buffer
- 7. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute
- 8. Envision 9.Envision 15 minutes
- 9. 10.Rinse in Buffer Buffer
- 10. Buffer Buffer Buffer Wash in Buffer x 3 Dip 10 times Dip 10 times 1 minute
- 11. DAB 11.DAB 10 minutes
- 12. 12.Rinse in DW DW d
- 13. Wash in DW 1 Minute
- 14. Hematoxylin 13.Hematoxylin 30 sec to 1 minute
- 15. 14.Rinse in Tap Water
- 16. 15.Coverslip with Aqeous medium