英文免疫组化.pptVIP

Immunohistochemistry Antibodies recognize proteins, peptides, or amines Localize presence of transmitters receptors in tissues Procedure Incubate tissue with Ab so it binds to antigen present in the tissue Wash tissue so only bound antibody remains Expose tissue with Ab to second Ab which recognizes 1st Ab 2nd Ab is conjugated to fluorescent chemical or enzyme system which forms a visible reaction product Major Goal Maximize signal positive staining Minimize background staining false positive staining Procedure Modification Add Neutravidin which will be attracted to biotinylated secondary Ab. Neutravidin will be conjugated to the fluorescent tag Alexa 488 green fluorescent tag or CY red fluorescent tag Ab with Specific Recognition Characteristics Primary Ab raised in rabbit Secondary Ab raised against rabbit serum in some other animal sheep, goat, or rodents Double Labeling For double labeling- primary Ab rasied in different animals rabbit and rats in case of double labeling secondary Ab have different materials conjugated anti-rabbit IgG conjugated with fluorescein and anti-rat IgG conjugated with HRP Detailed Procedure Tissue fixed Tissue cut into thinner sections 15uM Mounted on gelatin or poly-D-lysine sections Use of Triton X Use of serums directed against 2nd Ab , dry milk products, avidin, H2O2 Controls Best procedure- incubate primary Ab with antigen - antigen preabsorption Omit the primary Ab in the 1st incubation step Use a positive control-use tissue known to contain antigen of interest. High Background Staining 1. Endogenous peroxidase activity-not completely blocked. 2. Non-specific binding of protein to specimen-need for blockers. 3. Excessive application of tissue adhesive 4. Inadequate rinsing of slides 5. Improper antibody dilution 6. Drying out of tissue. 7. Overdevelopment of substrate. Possible Causes of Negative Staining Steps for staining not performed in correct sequence. Either primary or secondary antibody incuba