ICC/IHC实验指南
ICC/IHC Protocols
Protocol for the Preparation and Fluorescent IHC Staining of Frozen Tissue Sections
Enzymatic Protocol 2.0
Materials
Cytokine-specific Primary Antibodies unlabeled or biotinylated antigen-affinity purified polyclonal antibodies R&D Systems 'AF or BAF series or selected monoclonal antibodies.
Secondary Antibodies and Secondary Reagents
Enzymatic Protocol 1.0
Overview
Troubleshooting Guide: Immunohistochemistry
Technically, IHC and ICC are relatively simple and straightforward experimental methods. However, there are many variables which must be identified and optimized for each individual IHC/ICC study. Optimization of IHC/ICC may also require troubleshooting a variety of factors. The tables below highlight common IHC/ICC issues and provide appropriate experimental actions. For further assistance, please contact our technical service department.
Preparing Samples for IHC/ICC Experiments
Tissue and cell samples must be appropriately harvested and prepared for each IHC/ICC study. To facilitate the required incubation steps, whole tissues must be cut into ultra thin (5-10 µm) sections or cut into smaller pieces for whole mount IHC. For ICC experiments, cells must be attached to a microscope slide or coverslip before commencing the staining procedure. Sample preparation is also intimately linked to the method of fixation, which in turn is influenced by the desired detection technique (fluorescence versus chromogenic).
Preventing Non-specific Staining
Once tissue or cell samples have been appropriately prepared and fixed, the samples are ready to be stained. All IHC/ICC studies are dependent on specific antibody-epitope binding, which is governed by hydrophobic interactions, ionic interactions, hydrogen bonding, and other intermolecular forces. However, the same attractive forces can also result in non-specific staining, i.e. binding of the primary antibody to amino acids other than those within the desired epitope of the antigen. This is a common problem that occurs in IHC/ICC experiments.
Graphic Protocol for the Preparation of a Cell Smear for Non-adherent Cell ICC
The Importance of IHC/ICC Controls
Appropriate controls are critical for the accurate interpretation of IHC/ICC results. A satisfactory IHC/ICC experimental design produces results that demonstrate that the antigen is localized to the correct specialized tissues, cell types, or subcellular location. Optimization of fixation, blocking, antibody incubation, and antigen retrieval steps will generate a strong and specific signal. However, IHC/ICC experiments must include positive and negative controls to support the validity of staining and identify experimental artefacts.
Graphic Protocol for the Fluorescent ICC Staining of Cultured Cells on Coverslips
Protocol for Heat-Induced Epitope Retrieval (HIER)
Antigen retrieval can reveal epitopes masked during the preparation of tissues for staining. The protocol below describes a technique used by R&D Systems and can be used on both cryostat and paraffin-embedded sections. The protocol can be used with R&D Systems Acidic, Basic, or Universal Antigen Retrieval Reagents. Pretreatment with these solutions may induce a dramatic enhancement of immunoreactivity. However, they also have the potential to affect tissue morphology.