Paraffin section immunohistochemical staining experimental steps - Database & Sql Blog Articles

Paraffin section immunohistochemical staining experimental steps: 1. Paraffin sections are dewaxed to water: (The paraffin section should be placed at 60 ° C for 1 hour before dyeing). (1) Xylene I, II, each 10 minutes. (2) Gradient Alcohol: 100%, 2 minutes ® 95%, 2 minutes ® 80%, 2 minutes ® 70% 2 minutes. (3) Distilled water washing: 5 minutes, 2 times (on a shaker). 2. Hydrogen peroxide blocked the endogenous peroxidase: 3% H2O2 at room temperature for 10 minutes (protected from light). 3. Distilled water wash: 5 minutes, 2 times (on a shaker). 4. Antigen retrieval: Select the appropriate method based on the antigen to be detected. Attachment: Preparation of antigen repair solution (10mM pH 6.0 sodium citrate buffer) (1) Preparation of stock solution: Liquid A: trisodium citrate-2H2O 29.41g + distilled water 1000mlB solution: citric acid 21g + distilled water 1000ml (2) Preparation of working fluid: A solution 82ml + B solution 18ml + distilled water 900ml antigen repair method: (1) Pressure cooker treatment technology: sodium citrate buffer (10mM, PH6.0), submerged slices, covered with pot Cover, boil in the pressure cooker, and slowly cool after steaming for 3 minutes (use tap water to flush outside the pressure cooker to help cool). (2) Microwave treatment technology: use plastic slicing rack, placed in plastic or temperature-resistant glass container, submerged slice with sodium citrate buffer, select medium or high grade, 5 minutes; remove and replenish preheated sodium citrate Buffer; select medium or high, 5 minutes (. optimal temperature is 92 ~ 95 ° C) (3) enzyme digestion treatment: this slightly. Precautions for antigen retrieval: (1) The organization cannot do it. (2) The method of selecting an antigen to be repaired varies depending on the antibody. (3) This method is mainly used for 10% formalin-fixed, paraffin-embedded tissues. (4) PBS buffer is required in the process of antigen restoration to DAB color development. 5. PBS: 5 minutes, 2 times (on a shaker). 6. Normal serum occlusion: remove the slice from the dyeing cylinder, wipe the water on the back of the slice and the water around the front tissue of the slice (keeping the tissue moist), and add normal goat or rabbit serum (with the second antibody homologous animal serum) , 37 ° C, 15 minutes.