Immunohistochemistry (ABC method) on mouse brain slices
Immunohistochemistry (ABC method) on mouse brain slices
Understand the principles of immunohistochemistry experiments and be proficient in the experimental procedures. (Neurobiology Laboratory Handout; edited by Du Jizeng and Chen Xuequn; 2004.09)
Operation method
immunohistochemistry
Principle
A method of examining in situ antigenic or antibody components on cells and tissues by labeling visible displays on specific antigen-antibody reactions. The nature of the localization is observed under a light microscope, fluorescence microscope or electron microscope, and cellular in situ semi-quantitative measurements can also be performed using cell spectrophotometers, image analyzers, confocal microscopes, and so on. The distinctive feature of immunohistochemistry is its high specificity.
Materials and Instruments
Frozen sections Move 1. Remove the reagents for the experiment from the refrigerator at 4 degrees Celsius and equilibrate naturally to room temperature; Caveat 1. Endogenous biological activity and its elimination Biotin is a coenzyme, a carboxylate intermediate carrier produced in the metabolic process catalyzed by decarboxylase, carboxylase, etc., which will bind to the anti-biotin protein when applying the ABC method and produce non-specific staining. For tissues rich in endogenous biotin or biotin-like substances, before applying the ABC method, the tissues should be pre-activated with 0.01% antibiotic proteins and 0.01% biotin solution for about 20 minutes to eliminate endogenous biotin activity. PBS wash for 5 minutes should be applied after each action and replaced three times.In addition, recently there have been from streptavidin (streptavidin) due to its molecule does not contain oligosaccharides and isoelectric point close to neutral (6-6.5), so the non-specific adsorption is very low. At the same time, streptavidin can be combined with long arm biotin and peroxidase to form a complex, which is SABC (streptavidin-Biotin-peroxidase Complex) complex, using SABC instead of ABC for labeling, can improve the sensitivity (about 20 times higher than ABC) and reduce the non-specific coloring. SABC is now available in finished kits, and the procedure is basically the same as the ABC method, except that instead of labeling with ABC, 10 μl of each of solution A and solution B from the SABC kit is added to 1 ml of PBS (prepared before use, and added to the section immediately after preparation) and incubated at room temperature for 30 minutes. In addition, the incubation time for both primary and secondary antibodies can be shortened to less than 30 minutes, so the SABC method is a rapid, simple, sensitive and low non-specific coloring detection method. 2. The mutual affinity between biotin preparations varies greatly, so when applying ABC reagents, attention should be paid to the manufacturer and batch number, and prior prediction should be made for the purchased reagents to ensure the stability of the experimental results. 3. Tissue sections should always be kept moist during labeling. 4. Adding metal ions, such as nickel, to the DAB display solution to make the reaction product blue-black, and re-staining the nuclei with methyl green can enhance the contrast of positive staining and improve sensitivity. 5. The process of rinsing with PBS buffer should be as complete as possible, with no impurities left on the surface of the slice. 6. accurately proportional to the concentration of the antibody, because the concentration is too high or too low is not conducive to the progress of the reaction, in the titration of the antibody, to achieve a small and uniform amount. 7. DAB is a toxic reagent, try not to contaminate the surrounding environment, and try to avoid the decomposition of DAB in the process of color development. 8. Add each reagent quickly and accurately, so as not to dry the tablets in the reaction. 9. 4 degrees Celsius is the best temperature for preservation of ABC reagent, and it is reported that it can be preserved for two years and still get satisfactory results, while at -20 degrees Celsius, the biological activity will be destroyed in a short period of time. For more product details, please visit Aladdin Scientific website.
Primary antibody; secondary antibody; tertiary antibody (biotin-labeled horseradish peroxidase ovalbumin); PBS buffer; paraformaldehyde; DAB
Immuno wet kit; dropper; scissors; imported film (parafilm); 37 degrees incubator; pipette gun and tip; enzyme pen (PAP); light microscope
2. Remove brain slices from -20/-80 degree refrigerator and equilibrate naturally to room temperature.
3. draw a circle around the brain slice with an enzyme marker to prevent liquid from overflowing on the slide.
4. 4% paraformaldehyde was fixed for 30 min.
5. 0.01M PBS wash, 5 minX3 times.
6. 0.4% Triton-X100/0.01 M PBS wash for 10 min X 1 time.
7. 0.01 M PBS wash, 5 min X 3 times.
8. 0.3% H2O2/0.01 M PBS wash 10 min X 1 time.
9. 0.01 M PBS wash, 5 min X 3 times.
10. with 10% goat serum (1:10, diluted in 0.01 M PBS), left at 37 degrees Celsius in a thermostat for 30 min, usually at 40 ul/brain slice.
11. Pour off the closed serum, slightly dry, add primary antibody, and place in the refrigerator at 4 degrees overnight.
12. 0.01M PBS wash, 5 min X 3 times.
13. Add secondary antibody (1:200 dilution) and leave at 37 degrees Celsius for 1h, usually at 40 ul/brain slice.
14. 0.01M PBS wash, 5 min X 3 times.
15. Add secondary antibody (1:300 dilution) and place at 37 degrees Celsius for 1h, generally at 40 ul/brain slice.
16. 0.01M PBS wash, 5 min X 3 times.
17. DAB color development (DAB configuration: 1 ml of water, add 1 drop of liquid A, mix well, add 1 drop of liquid B, then 1 drop of liquid C), color development about 10 min.