Experimental reagent
Liquid nitrogen, dry ice, 1% gelatin, 4% paraformaldehyde, PBS, 1% NP-40 in PBS, 3% BSA / PBS diluted antibody, horseradish peroxidase labeled secondary antibody, DAB / metal salt reagent , 3% hydrogen peroxide, 0.3% (W / V) nickel chloride stock solution, Harris hematoxylin, ethanol
experiment apparatus
Slides, Whatman 1 # filter paper, low power optical microscope
Experimental Materials
Undamaged tissue
Experimental procedure
1. Cut the intact tissue into small samples, about lcmXIcmX0.4cm.
2. Place the specimen on one end of the card. Mark the card and immerse the end with the specimen in liquid nitrogen. After 60s, remove the specimen and place it on dry ice. Trim the card to a size slightly larger than the tissue block, and transfer it to a pre-cooled (-70 ° C) labeled vial.
3. Before slicing, coat clean slides with 1% gelatin. Heat to 50 ° C to dissolve gelatin in water, cool and add 0.02% sodium azide. Immerse the slide in the gelatin solution for 30s, take it out, and let it dry naturally.
4. Prepare cryosections with a cryostat according to the instruction manual of the instrument. The slice thickness is usually between 5-10gm. Collect slides with coated slides.
5. Let the slices dry naturally. Immerse in freshly prepared 4% paraformaldehyde for 2 minutes.
6. Wash several times with PBS and put 5rain in PBS containing 1% NP-40. Wash several times with PBS.
7. Place slides with tissue sections in a wet box. Add an appropriate dilution of primary antibody and dilute the antibody with a protein-containing solution such as 3% BSA / PBS.
The monoclonal antibody is preferably selected from the culture supernatant of hybridoma cells (specific antibody concentration is 20-50gg / m1). Mouse ascites, purified monoclonal antibodies and polyclonal antibodies, and crude polyclonal antisera should be tested for dilution so that the concentration of specific antibodies is between 0.1 and 10 ug / m1. If the concentration of the specific antibody is unknown, prepare and test different dilutions of the initial preparation (1:10, 1: 100, 1: 1000, and 1:10 000).
8. Incubate the slides in a wet box at room temperature for at least 60 minutes. For some reactions, the incubation time can be extended to 24h to increase its sensitivity.
9. Wash 3 times with PBS for more than 5 minutes each time.
10. Add a secondary antibody labeled with horseradish peroxidase (specific for the primary antibody). These reagents can be purchased from different distributors. If the exact amount of secondary antibody is unknown, test the secondary antibody diluted 1: 50-1: 1000. Dilute with protein-containing PBS, such as 3% BSA / PBS.
11. Place the sample in a wet box and incubate at room temperature for 30 minutes.
12. Wash three times with PBS for more than 5 minutes each time.
13. Prepare DAB / metal salt reagent. 6 mg DAB was dissolved in 9 ml 0.05 mol / L Tris buffer (pH 7.6). Add lm [0.3% (W / V) nickel chloride stock solution (can be replaced with the same concentration of cobalt chloride). Add 0.1ml of 3% hydrogen peroxide. If precipitation occurs, filter with Whatman 1 # filter paper (or similar).
14. Add the above solution to the specimen. Observe under a low power optical microscope. When sufficient brown / black precipitate is formed, rinse with water to stop the reaction. Usually this reaction process takes about 1-20min.
15. Add a few drops of Harris hematoxylin. Incubate for about 5 minutes. The length of time depends on the intensity of dyeing.
16. Rinse gently with water.
17. Dehydrate the specimens sequentially with ethanol at all levels. Incubate twice in 75% ethanol, 3 minutes each time, 2 times in 95% ethanol, 3 minutes each time, 2 times in 100% ethanol, 3 minutes each time. Naturally dry.
18. Add a small drop of DPX to the specimen. Carefully place a cover slip (1 #) on it to avoid air bubbles. Use paper towels to remove excess liquid. DPX can solidify quickly, and the sample can be observed and photographed.
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