Processing of Cells and Tissues for Electron Microscopy

3.2.1. Detection of Cannabinoid Receptors by Immunoperoxidase Staining

3.2.1.1. Perfusion of Mouse/Rat and Removal of Brain With 4%

Paraformaldehyde, 0.1% Purified Glutaraldehyde Fixative in 0.1 M

Phosphate Buffer

1. Prepare separate flasks containing cold (4°C) saline (0.9%) and room-temperature fixative solution.

2. Prepare peristaltic pump, Tygon tubing, and perfusion instruments according to manufacturer's instructions. Arrange such that saline will be drawn through the tube first by configuring the tubing with a valve to allow for selection of solution to be drawn.

3. Attach Tygon tubing primed with saline to a blunt 15 G hypodermic needle.

4. Administer a lethal dose of anesthesia to mouse or rat. Make certain that the animal is unresponsive before proceeding. Lack of responsiveness can be determined by pinching the animal's foot.

5. Make an incision through the abdomen just below the rib cage and expose the diaphragm. Make incision through the diaphragm and expose the heart.

6. Open the thoracic cavity with two horizontal cuts through the rib cage on either side of the heart. Clamp the sternum with a hemostat and fold the cut rib flap head-ward to expose the heart

7. Make a small incision at the bottom apex of the left ventricle. Insert a blunt 13 G hypodermic needle upward through the ventricle past the aortic valve so that it can be visualized at approx 5 mm inside the ascending aorta. Clamp the needle in place with a hemostat across the ventricle.

8. Begin perfusion by slowly (i.e., 20-40 mL/min) introducing perfusion prewash solution. Immediately after the peristaltic pump starts pumping saline, cut the right atrium to allow for an escape route for blood and perfusion fluid. Keep saline on ice so as to minimize coagulation of blood

9. Continue to perfuse with perfusion prewash solution (approx 40 mL/min) until the effluent runs clear. Perfusion may require up to 500 mL of saline.

10. Stop the peristaltic pump and begin the flow of fixative. Perfuse at approx 20 mL/min such that approx 500 mL of fixative is perfused over a 10- to 20-min period.

11. Remove the brain and immerse it overnight in 0.1 M sodium phosphate buffer.

3.2.1.2. Sectioning of Mouse/Rat Brain and Incubation of Brain

Sections With the Primary Anti-Cannabinoid Receptor Antibody

1. Cut brain (embedded in 4% agar, 50-100 |im sections) using a Vibratome according to the manufacturer's instructions. Place sections into glass vials with caps filled with 0.1 M sodium phosphate buffer. Note: Sections can be transferred from one solution to another with a camel hair's brush. Subsequent steps can be performed by treating sections within the glass vials under gentle agitation by placing vials on a rotator mixer.

2. Rinse sections with PBS three times (10 min each) at room temperature.

3. Treat sections with 0.1% phenylhydrazine (endogenous peroxidase activity inhibitor) at room temperature for 30 min.

4. Rinse sections with PBS three times (10 min each) at room temperature.

5. Preincubate sections (30 min) in 5 mL of blocking solution of PBS supplemented with 4% NGS, 0.3% Triton X-100, 0.1 M lysine (0.146 g/10 mL), and 1% BSA

7. Incubate sections overnight in a refrigerator (4°C) on a shaker with affinity-purified primary rabbit anti-cannabinoid receptor antibody (see Table 1). Use the affinity-purified antibody at a 1:1000-1:5000 dilution of a stock antibody preparation (1 mg/mL) in a final volume of 5 mL PBS supplemented with 1% NGS. Note: For immunoelectron microscopy it is recommended that affinity-purified antibody be used (see Note 1).

3.2.1.3. Incubation of Vibratome Sections in Secondary Antibody, Immunoperoxidase Development, and Initial Screening by Light Microscopy

1. Rinse sections three times (10 min each) in 1% NGS.

2. Incubate sections (1 h at room temperature) in biotinylated goat anti-rabbit IgG (1 mg/mL stock solution). Prepare by adding 50 |L per 10 ml 1% NGS.

3. Rinse sections three times (10 min each) in PBS. Note: At this time prepare the ABC solution (Vector Laboratories, Burlingame, CA). Add 50 | L of solution A (Avidin DH) to 10 mL PBS. Then add 50 |L of solution B (biotinylated horseradish peroxidase H). Mix immediately. Let stand for 30 min before use.

4. Incubate tissue sections in ABC solution for 1 h at room temperature (see Note 2).

5 Rinse sections three times (10 min each) in PBS.

6. Rinse sections twice (10 min each) in 0.1 M sodium phosphate buffer.

7. Preincubate sections (10 min) in DAB substrate with nickel (Ni) solution without hydrogen peroxide (H2O2). To prepare, use the DAB substrate kit (Vector Laboratories, Burlingame, CA). Add two drops buffer stock to 5 mL of distilled water and mix. Add four drops DAB stock solution and mix. Add two drops of nickel solution and mix.

8. Incubate sections in DAB substrate with nickel plus H2O2. Prepare from the DAB substrate kit (Vector Laboratories) as in step 7 and add two drops of H2O2 solution. Mix well. Note: Determination of the optimal time for development of reaction product for electron microscopy assessment is achieved best by monitoring the development process under a light microscope. Ideally, sections are obtained for three development periods. The first of these is allowed to over-develop so as to allow for definition of which sites within the tissue section are positive for immunoproduct. The second section, then, is monitored for development just to the point where product is first observed. Finally, the third section is allowed to develop for a time period which is just under that obtained for the second section. The latter two sections are examined under the electron microscope. Using this approach, it is possible to obtain sections for observation under the electron micro scope for which diffusion of immunoelectron dense product from antigenic sites is limited.

9. Rinse sections (10 min) in 0.1 M sodium phosphate buffer.

10. If required, rinse sections (5 min) with fixative solution to clear background.

11. Rinse sections twice (10 min each) in 0. 1 M sodium phosphate buffer.

12. Mount a representative number of tissue sections out of 0.1 M sodium phosphate buffer for initial assessment by light microscopy. For light microscopy, proceed to step 13. For electron microscopy, proceed to Subheading 3.2.1.4.

13. Dehydrate and coverslip and screen by light microscopy according to the following schedule:.

d. Absolute ethanol (2-3 min).

h. Mount in Permount Organic Medium (Fisher Scientific, Pittsburgh, PA). Allow Permount to harden on a slide warmer.

3.2.1.4. En Bloc Staining of Tissue With Uranyl Acetate and Processing for Transmission Electron Microscopy

1. Place vials on ice in a chemical fume hood. Remove buffer and add 300 |L of cold (4°C) 1% osmium solution. Snap on cap of vial and incubate for 1 h at 4°C with gentle agitation on a rotary mixer. Note: OsO4 is highly toxic and volatile. Perform all steps in a chemical hood. Do not breathe in vapors and do not allow contact with skin. Dispose of all used solutions in a chemical waste bottle that is maintained in the chemical hood. Dispose of all waste using procedures mandated by your institutional biohazardous waste/safety officer.

2. After postfixation with osmium tetroxide, wash tissue sections thoroughly in cold (4°C) sodium hydrogen maleate-NaOH buffer, 0.05 M, pH 5.2 (three changes, 5 min each).

3. Immerse tissue sections in 0.5% uranyl acetate (Sigma-Aldrich, St. Louis, MO) dissolved in 0.05 M sodium hydrogen maleate-NaOH buffer, pH 6.0 (50 mg/10 mL) for 2 h at 4°C in the dark.

4. Rinse tissue sections in cold (4°C) sodium hydrogen maleate-NaOH buffer, 0.05 M, pH 5.2 (two changes, 5 min each).

5. Proceed with dehydration and embedding according to the schedule below. Note: All steps are performed on ice with frequent agitation. Tissue sections are placed in glass vials with caps, which, in turn, are placed in a rotary mixer (Penetron Mark IIIB, Sunkay Laboratories Inc., Tokyo, Japan).

Fig. 2. Brain serial cross-sections which have been subjected to immunoperoxidase preembedding immunoperoxidase reaction for localization of the CB1 receptor and oriented on a glass microscope slide coated with liquid release agent. An area of interest has been identified as having intense labeling under the light microscope and has been excised (arrow) using a sharpened cork borer for mounting on resin stubs.

Fig. 2. Brain serial cross-sections which have been subjected to immunoperoxidase preembedding immunoperoxidase reaction for localization of the CB1 receptor and oriented on a glass microscope slide coated with liquid release agent. An area of interest has been identified as having intense labeling under the light microscope and has been excised (arrow) using a sharpened cork borer for mounting on resin stubs.

e. 100% (absolute) methanol (two changes of 10 min each).

f. Propylene oxide (two changes of 10 min each).

g. Propylene oxide, TAAB resin (1:1 mixture) (1 h on a rotary mixer).

h. Straight TAAB resin for 4-8 h on rotatory shaker. During the last hour, remove caps from tissue vials and place under vacuum in a desiccator to remove air.

6. Using an applicator stick (broken to make flat tapered tip), transfer each section with a small amount of TAAB to a glass microscope slide coated with liquid release agent (Electron Microscopy Sciences, Fort Washington, PA). The size of the sections will determine how many can be arranged on a single glass slide. Sections should be properly oriented with at least 5-mm separation (Fig. 2). Place another coated glass microscope slide over each group of sections. Do not overlap the sides and ends of the two slides. Press lightly over each section to spread resin and clamp slides together with clothespins. Place slides with sections on flat surface and polymerize in oven at 60°C for 48-72 h to harden resin.

7. Pry the two slides apart carefully using a single-edged razor blade. Identify the area of interest or intense labeling under the light microscope. Under a dissecting microscope, use a scalpel or a sharpened cork borer (approx 2-3 mm in diameter) and cut out area of interest for mounting on resin stubs (Fig. 2, arrow).

8. Place embedded tissue sample on glass microscope slide coated with liquid release agent. Place a drop of liquid TAAB resin onto one flat end of a hardened resin stub. Place same end of stub on top of tissue sample. Place a small drop of resin on the other end of the stub and affix a small piece of paper with a label identifying the sample. A circular piece of paper resulting from using a hole puncher will suffice.

9. Place slide with stubbed tissue in a 60°C oven for 2-3 d.

10. Trim the surface of the tissue block and subject to thin-sectioning on an ultramicrotome.

11. Examine in an electron microscope. Figure 3 illustrates the comparative application of light and electron microscopy for the localization of the CB1 cannabinoid receptor in rat brain. Figure 4 illustrates high-resolution localization of the CB1 receptor within rat brain.

3.2.2. Detection of Cannabinoid Receptors by Immunogold Labeling

3.2.2.1. Preparation of Tissue ( Note: All steps are performed at 4°C

unless otherwise indicated)

1. Immerse 2-mm3 pieces of tissue in fixative consisting of 4% paraformaldehyde and 0.25% glutaraldehyde in 0.1 M sodium cacodylate/HCl buffer for 3-4 h.

2. Rinse tissue in 0.1 M sodium cacodylate/HCl buffer. Use four to five changes over a 4-h minimum. It is preferable to leave tissue samples overnight in buffer (on a rotator in a cold room maintained at 4-10°C).

3. Dehydrate tissue samples in a graded series of ethanol: 50% (20 min), 75% (20 min), and 100% (three times, 20 min each with rotation).

3.2.2.2. Embedding in Resin

1. Remove the final absolute ethanol and add LR gold resin (Polysciences Inc., Warrington, PA). Note: Depending on the size of the sample, allow to infiltrate in resin for at least 5 d (up to 7 d for larger samples). Change the resin every 2 d. For the last two changes of the LR gold include 0.1% benzoin methyl ether (the light-activated catalyst).

2. Polymerization: Pipet the samples into gelatin capsules so that they are filled with LR Gold resin (supplemented with 0.1% benzoin methyl ether) and replace the cap on the capsule because the light-activated polymerization is anaerobic. Place the gelatin capsules in a 96-well microtiter plate on a plate of glass held approx 25 cm above a near ultraviolet lamp set-up in a cold room (4°C). Allow to polymerize for 48 h.

Fig. 3. Comparative light and electron microscopy for the localization of the CBi cannabinoid receptor in the rat cerebellum. (A) Light microscopy immunoperoxidase staining showing immunoreactive product in an area "cradling" or apposed (arrow) to the basal soma of a Purkinje cell (PC). (B) Transmission electron micrograph illustrating similar immunoreactivity for the CBi receptor (arrows) in areas apposed to the basal portion of a nonimmunoreactive Purkinje cell (PC) body. Note absence of reaction product from Purkinje cell proximal dendrite (d). Scale bars: A, 10 |im; B, 5 |im. O, oligodendrocyte.

Fig. 4. Transmission electron micrograph illustrating a small immunoperoxidase-reactive synaptic ending (s) establishing contact (arrow) with a spine-like profile (sp). Note the presence of dense immunoreactive product associated with the presynaptic membrane and encompassing adjacent synaptic vesicles. Scale bar, 0.25 |im.

Fig. 4. Transmission electron micrograph illustrating a small immunoperoxidase-reactive synaptic ending (s) establishing contact (arrow) with a spine-like profile (sp). Note the presence of dense immunoreactive product associated with the presynaptic membrane and encompassing adjacent synaptic vesicles. Scale bar, 0.25 |im.

3. Sectioning: Ultrathin sections of LR gold-embedded specimens are cut as for standard (epoxy) electron microscopy resins. Cut sections fresh for any immunolabel-ing experiment. Note: Collect sections on nickel grids (300 mesh). Grids may be used with or without Formvar coating.

3.2.2.3. Immunolabeling of Plastic Sections for Cannabinoid Receptors

1. For all incubations place grids on 20-|L droplets on Parafilm within a Petri dish. Rinses in buffer are performed by floating grids section side down on droplets. Transfer grids from one solution to another with fine forceps using care to not to wet the opposite face of the grid that is being labeled.

2. Blocking step: Incubate grids in MPBS containing 1% BSA for 30 min at room temperature. If immune tissue or cells are being examined, add 1% NGS to the blocking buffer in order to block Fc receptors, which are present on the surface of some immune cells.

3. Incubate in affinity-purified rabbit anti-cannabinoid receptor IgG overnight at 4°C (in a humidified chamber). A first immunolabeling should include a titration series of the specific affinity-purified antibody at dilutions of 1:5-1:20 to establish the optimal dilution. This titration series is required to optimize the signal-to-noise ratio of immunolabeling. Concurrent incubations should be carried out in nonimmune rabbit IgG (at similar dilutions) from the primary antibody donor species to serve as negative controls.

4. Rinse the grids in MPBS (three times, 2 min each).

5. Incubate the grids in goat anti-rabbit IgG-polygold (10 nm). Dilute the gold probe 1:10 in MPBS immediately before use. Incubate for 1 h at room temperature.

6. Rinse the grids thoroughly in buffer (three times, 10 min each).

7. Rinse the grids in double glass-distilled water (four times, 3 min each).

3.2.2.4. Staining of Sections on Grids for Electron Microscopy

1. Stain sections on grids for 5-10 min in 0.5% aqueous uranyl acetate. Cover from light to prevent precipitation.

2. Rinse the grids by dipping (10-20 dips) in reagent quality water.

3. Stain sections on grids with lead citrate (1-2 min). Stain in a plastic dish with NaOH pellets to prevent CO2 from causing precipitation. Lead citrate should be dispensed from the top of the solution. Do not breathe on the lead citrate at any stage, as insoluble lead carbonate will form, which produces dense spots over the sections.

4. Rinse grids by dipping (10-20 dips) in reagent grade water. Note: LR Gold resin is hydrophilic and takes up the uranyl and lead stains considerably more rapidly than the standard epoxy resins.

5. Dry overnight.

6. Examine grids in the transmission electron microscope. Figure 5 illustrates the application of the immunogold method at the electron microscopy level for the identification of the human CB2 as expressed in the envelope of a baculovirus construct (13).

Fig. 5. Immunogold labeling for the demonstration of the human CB2 cannabinoid receptor as expressed on the envelope of a baculovirus CB2 expression vector in insect cells. (A) Intense immunogold label surrounds the envelope (arrow) of the baculovirus construct. (B) Control P-galactosidase recombinant baculovirus envelopes show no immunolabeling for the CB2 cannabinoid receptor. Scale bars, A and B, 0.2 |im.

Fig. 5. Immunogold labeling for the demonstration of the human CB2 cannabinoid receptor as expressed on the envelope of a baculovirus CB2 expression vector in insect cells. (A) Intense immunogold label surrounds the envelope (arrow) of the baculovirus construct. (B) Control P-galactosidase recombinant baculovirus envelopes show no immunolabeling for the CB2 cannabinoid receptor. Scale bars, A and B, 0.2 |im.

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