实验概要
Immunohistochemistry is a classic technique used for the localization of antigenic target molecules in tissue. The method exploits the principle that the target antigen is recognized by specific antibody and is visualized using different detection systems. The subject of this chapter is simultaneous immunohistochemical detection of protein antigens and proliferation marker BrdU in the developing tooth.
实验原理
Immunohistochemistry (IHC) is currently one of the most popular histological techniques, which is widely used to recognize and detect antigens in different tissues. The principle behind the IHC is specific binding of antibodies to target antigenic epitopes and their subsequent detection (1, 2).
The method was introduced in 1941, when identification of tissue antigens using a direct fluorescence protocol was described (3). Since then, many monoclonal and polyclonal primary antibodies have been developed. The primary antibody can be directly conjugated to a fluorophore or an enzyme, which can catalyze color reaction. Alternatively, the primary antibody can be recognized with labeled species-specific secondary antibodies and detected indirectly. In some systems, such as avidin–biotin complex (ABC), tertiary antibodies are used. Secondary and tertiary antibodies allow amplification of the signal, and subsequently more sensitive detection of antigens (2, 4). In this chapter, the primary antibodies are detected by secondary antibodies conjugated to fluorescent cyanine dyes (e.g., Cy-2, Cy-3, and Cy-5) (5).
In 1981, immunostaining based on the use of antibodies against 5-bromo-2′-deoxyuridine (BrdU) was first used to detect cells in S (synthetic) phase of cell cycle. BrdU is a thymidine analog, which can be incorporated into the newly synthesized DNA of replicating cell and recognized by anti-BrdU-specific antibodies. Binding of the antibodies requires DNA denaturation, achieved by heat treatment or low pH. BrdU immunostaining and subsequent quantification of cells positive for BrdU allow determination of proliferation rates and cell cycle kinetics in different tissues (6–8).
In many cases, two or more antigens can be detected in the same tissue specimen at the same time. Multicolor immunohistochemistry that uses double and triple labeling is possible and can be used to study co-localization of several molecules. When two or more antigens are present in the same compartment, their co-localization is marked by a mixed color (9, 10).
In this chapter, we present a technique that has proven effective in the detection of protein antigens and BrdU in the frozen sections of embryonic mouse tooth.
主要试剂
1. In Vivo Labeling of Murine Cells with BrdU
1) 5BrdU.
2) Phosphate-buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.76 mM KH2PO4 in dH2O, pH 7.4 (with HCl).
3) BrdU solution: Dissolve 5 mg of BrdU in 1 ml of PBS. Store at −20°C.
2. Preparation of the Frozen Sections
1) PBS.
2) 4% Paraformaldehyde in PBS (see Note 1).
3) 30% Sucrose in PBS.
4) OCT compound.
5) Ethanol.
6) Dry ice.
3. Immunostaining of Protein Antigens
1) PBS.
2) Methanol.
3) Fetal bovine serum: Heat inactivate at 60°C for 30 min; store in aliquots at −20°C.
4) Donkey serum: Heat inactivate at 60°C for 30 min; store in aliquots at −20°C.
5) Triton X-100.
6) Blocking buffer: 0.3% blocking reagent (Roche), 5% heatinactivated fetal bovine serum, 5% heat-inactivated donkey serum, and 0.1% Triton X-100 in PBS. Filter through fi lter paper and then through 0.2- μ m fi lter. Store in aliquots at −20°C.
7) Primary antibody against an antigenic protein of interest.
4. Post-antibody Washes and BrdU Immunostaining
1) PBS.
2) PBST: PBS containing 0.1% Tween 20.
3) Blocking buffer.
4) Species-specific cyanine-labeled secondary antibodies, which bind primary antibodies against antigenic protein.
5) 4% Paraformaldehyde in PBS (see Note 1).
6) 2 N HCl in dH2O.
7) Boric acid.
8) 0.1 M borate buffer. Adjust the pH to 8.5 with 1 M NaOH.
9) Anti-BrdU antibodies.
5. Post-BrdUImmunostaining Washes
1) PBST.
2) Blocking buffer.
3) Para film M.
4) Species-speci fi c cyanine-labeled secondary antibodies, which bind anti-BrdU antibodies.
5) 100, 95, 70, and 50% ethanol in dH2O.
6) Xylene.
7) DPX mounting medium.
主要设备
1. In Vivo Labeling of Murine Cells with BrdU
1) 1.5-ml microfuge tubes.
2) 1-ml syringes.
2. Preparation of the Frozen Sections
1) 10-cm Petri dish.
2) Dissection microscope.
3) Dissection tools: Watchmaker’s forceps and razor blades.
4) 24- and 48-well plates.
5) Disposable embedding molds.
6) Cryostat (Leica), knife holder, glass anti-roll guide, disposable microtome knives, and specimen discs (see Note 2).
7) Micro slides superfrost plus.
8) Hot plate.
3. Immunostaining of Protein Antigens
1) Histology slide tray.
2) Para film M.
3) Filter paper.
4) 0.2- um filters.
4. Post-antibody Washes and BrdU Immunostaining
1) Histology slide tray.
2) Incubator.
3) Para film M.
5. Post-BrdUImmunostaining Washes
1) Histology slide tray.
2) Slide rack.
3) Microcover glasses.
实验步骤
1. In Vivo Labeling of Mouse Cells with BrdU
Carry out all procedures at room temperature unless otherwise speci fied.
1) Inject 1ul of BrdU solution per 1-g body weight into timedpregnant mouse (see Note 3).
2) Wait for 2 h before euthanize the animal (see Note 4).
2. Preparation of the Frozen Sections
1) Dissect embryos in ice-cold PBS in a Petri dish under dissection microscope, if necessary.
2) Remove heads and transfer them into 24- or 48-well plates, wash in ice-cold PBS (see Note 5).
3) Fix heads in 4% paraformaldehyde in PBS at 4°C for 1–2 h (see Note 6).
4) Wash heads in PBS at 4°C overnight.
5) Incubate heads in 30% sucrose in PBS at 4°C for 1–2 days (see Note 7).
6) Dip heads into OCT for 1 min.
7) Transfer heads to an embedding mold containing OCT. Orient heads as desired (see Note 8) and freeze in ethanol, which contains dry ice. Store frozen specimen blocks at −80°C for several months.
8) Attach block to the specimen disc and cut 8- to 20- um sections at −20°C in the cryostat (see Note 2). Thaw mount sections at room-temperature micro slides.
9) Dry slides on a 40°C hot plate for 30–60 min. Slides can be placed in a slide box and stored at −80°C for several days.
3. Immunostaining of Protein Antigens
Carry out all procedures in a histology slide tray unless otherwise specified.
1) Wash sections 3× with PBS, 10 min each time (to remove OCT).
2) Rinse sections with cold methanol at −20°C for 2–3 min (see Note 9).
3) Add 200 μl per slide of blocking buffer. Cover the slides with Para film coverslips carefully and incubate for 1h.
4) Carefully remove Para film coverslips and excess buffer from slides. Add 120 ul per slide of primary antibodies against antigenic protein diluted in blocking buffer. Cover the slides with Para film coverslips carefully. Incubate the slides at 4°C overnight (see Note 10).
4. Post-antibody Washes and Immunostaining of BrdU
Carry out all procedures in a histology slide tray unless otherwise speci fi ed.
1) Wash sections 3× with PBST, 10 min each time.
2) Add 120 μl per slide of blocking buffer with species-specific cyanine-labeled secondary antibodies, which bind primary antibodies against antigenic protein. Cover the slides with Para fi lm coverslips carefully. Incubate the slides for 2 h (see Note 10).
3) Wash sections 3× with PBST, 10 min each time.
4) Fix sections with 4% paraformaldehyde in PBS for 5 min.
5) Wash sections 3× with PBS, 5 min each time.
6) Transfer the slide rack to air incubator at 37°C. Add 2 N HCl and incubate the slides for 30 min.
7) Wash sections with 0.1 M borate buffer, pH 8.5, for 15 min.
8) Wash sections 3× with PBS, 5 min each time.
9) Add 200 μl per slide of blocking buffer. Cover the slides with Para fi lm coverslips carefully and incubate for 1 h.
10) Carefully remove Para film coverslips and excess buffer from the slides. Add 120 μl perslide of anti-BrdU antibodies diluted in blocking buffer. Cover the slides with Parafilm coverslips carefully. Incubate the slides at 4°C overnight (see Note 10).
5. Post-BrdUImmunostaining Washes
Carry out all procedures in a histology slide tray unless otherwise speci fi ed.
1) Wash sections 3× with PBST, 10 min each time.
2) Add 120 μl per slide of blocking buffer with species-speci fi ccyanine-labeled secondary antibodies, which bind anti-BrdU. Cover the slides with Para film coverslips carefully. Incubate the slides for 2 h (see Note 10).
3) Wash sections 3× with PBST, 10 min each time.
4) Transfer the slides to a slide rack. Wash the sections 2× with dH2O, 3 min each time.
5) Dehydrate sections 1× in 50, 70, and 95% ethanol in water, and 2× in 100% ethanol, 3min each time.
6) Incubate sections 2× in xylene for 3 min (see Note 11).
7) Mount slides with DPX and apply micro cover glasses, being careful not to trap any air bubbles. Let slides dry overnight (see Note 11).
注意事项
1. Make fresh 4% paraformaldehyde each time. Preparation should be carried out inside a fume hood. Store it at 4°C for up to 1 week.
2. See manufacturer’s manual for more details.
3. Intraperitoneal injections are recommended for mice.
4. Incorporation of BrdU can be detected in thymus and bone marrow in as little as 1 h post injection and 2 h in tooth. At 24 h post injection, BrdU can be detected in most tissues.
5. Make sure that most of blood is washed away (blood inhibits fi xation). Heads of older embryos can be cut by in half sagittaly at midline using razor blade. Each half can be embedded separately.
6. The fi xation timing should be optimized for each tissue individually, for example 2 h for E18.5 heads, 1 h 30 min for E16.5 heads, 1 h 15 min for E14.5 heads, etc.
7. Tissue has to sink in 30% sucrose in PBS.
8. Standard orientation planes for sections include sagittal, transverse, and frontal.
9. Permeabilization should only be required for intracellular epitopes when the antibody requires access to the inside of the cell to detect the protein. However, it will also be required for detection of transmembrane membrane proteins if the epitope is in the cytoplasmic region. There are a wide variety of detergents to choose from, and they differ in their ef fi ciency to extract membranes. The type of detergent and time for the permeabilization will require optimization for the tissue of interest.
10. Correct dilutions will contribute to the quality of staining if they are prepared accurately and consistently. Often, a manufacturer recommends dilution ranges compatible with other variables, such as method, incubation time, and temperature. If this information is not provided, optimal working dilution of the antibodies must be determined by titration.
11. The procedure should be carried out inside a fume hood.