High-Resolution Imaging of Neurotransmitter Receptors at Retinal Synapses

Samenvatting

Source: Zhang, J., et al. High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses. J. Vis. Exp. (2016)

This video demonstrates an electron microscopy technique for visualizing neurotransmitter receptor localization in retinal synapses. The retinal sections contain retinal ganglion cells that exhibit membrane-bound neurotransmitter receptors and are labeled with cholera toxin subunit B (CTB). Upon labeling the markers with gold nanoparticle-conjugated antibodies and counterstaining the section with heavy metals, the RGC receptors are visualized under a microscope.

Protocol

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.

1. Retinal Tissue Fixation

1. Assemble the following materials and tools: A dissecting microscope, 2 forceps with very fine tips, scissors, cellulose filter paper, plastic pipette and a microscope slide.
2. Anesthetize the rat in a closed chamber with 2.0 ml halothane (an inhalant anesthetic). Determine adequate anesthetization by these methods: lack of withdrawal of rear paw after toe pinch, or lack of blink reflex. Then decapitate immediately with guillotine. Remove the eyes with a pair of iris scissors and place in a glass dish containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB) at pH 7.4.
3. Using the dissecting microscope, remove the cornea by cutting off the front of the eyeball. Remove the lens and vitreous from the inner retinal surface with forceps.
4. Peel the sclera with the two forceps until the retina is isolated from the eyecup.
5. Cut the retina immediately into 100 - 200 µm-thick strips with a razor, and subject to pH-shift fixation.
6. Fix retina strips in 4% paraformaldehyde in 0.1 M PB at pH 6.0 for 20 - 30 min and then in 4% paraformaldehyde plus 0.01% glutaraldehyde at pH 10.5 for 10 - 20 min at room temperature (RT).
7. After several washes in PB with 0.15 mM CaCl₂ (pH 7.4 at 4°C), cryoprotect the retinal strips with glycerol (60 min each in 10%, 20%, 30%, then overnight [O/N] in 30%) in 0.1 M PB prior to freeze substitution.

2. Freeze-substitution

NOTE: This freeze-substitution method is modified from an earlier published protocol. Also, it is crucial that the instruments are very cold (wear gloves); otherwise, the tissue may thaw partially when touched with the instruments. All of these steps are done within the automatic freeze-substitution (AFS) chamber and the instruments are never allowed to move above the rim of the chamber. Similarly, proper cooling of all chemicals used in the AFS is necessary.

1. Plunge-freeze the retinal strips in liquid propane at -184°C in an electron microscope (EM) cryopreservation instrument (CPC). Using a fine brush, place the samples on small pieces of double-stick tape attached to the metal stubs that go on the end of the plunging rods (wick off extra buffer using the brush).
2. Plunge the rods into the liquid propane and keep them there for about 5 sec, and then transfer to the automatic freeze-substitution instrument (AFS) using a small transport chamber that is filled with liquid nitrogen (LN₂ cooled cryo-transfer container).
3. After placing the frozen sample and instruments (forceps and scalpel) into the AFS, cool the instruments in the chamber for several minutes before touching the tissue, or cool them by placing the tips of the instruments for a few seconds into the small transport chamber (filled with liquid nitrogen (LN₂ cooled cryo-transfer container)).
4. Once in the AFS, remove the sample and tape from the stub using a fine scalpel. Also, keep the nitrogen gas flow control, TF (TF is described as a 'regulator control for LN₂ vaporiser') open during these procedures.
5. Prior to placing the frozen tissue into the flat-embedding holders (i.e., before setting up the AFS), cut a thin circle from a clear plastic sheet and place it into the bottom of the holder to line the bottom of each well. This allows relatively easier removal of the polymerized specimen blocks when finished.
   NOTE: Previously, we used an alternative method to the flat embedding holders, and employing double Reichert+gelatin capsules.
6. Use the following automatic sequence in the AFS (using instrument terminology): T1 = -90°C for 32 hr, S1 = increase temperature by 4°C/hr (11.3 hr), T2 = -45°C for 50 hr, S2 = increase temperature by 5°C/hr (9 hr), T3 = 0°C for 40 hr (total = 142.3 hr). It may take 2 - 4 hr to place samples in the AFS (at -90°C) prior to starting the timed sequence.
7. Immerse the frozen sections in 1.5% uranyl acetate in methanol at -90°C for >32 hr in the AFS. Place two pieces of tissues (typically) in each of the seven wells in the flat-embedding aluminum holder (three fit into the AFS).
   1. Place the tissue+tape into the uranyl acetate/methanol in the wells and remove the tape later, just prior to beginning embedding medium (such as Lowicryl HM20) infiltration, if it is too difficult to remove the tissue from the tape.
8. Then increase the temperature stepwise to -45°C (+4°C per hr; in the automatic sequence).
9. Wash the samples three times in precooled methanol, by using a fine-tipped plastic pipette to remove the old solution from each flat-embedding holder, and then using another standard plastic pipette to add the precooled fresh methanol.
10. Then, using the same method, infiltrate the samples progressively with low temperature embedding resins such as embedding medium (HM20/methanol at 1:1 and 2:1, each for 2 hr, followed by pure resin for 2 hr and then change the resins and keep O/N).
11. Change the resin again the next day, adjusting the level of the resin to reach just to the top edge of the wells.
12. Polymerize the samples (-45°C to 0°C in automatic sequence; +5°C per hr) with ultraviolet (UV) light for 40 hr.
13. Then remove the samples from the AFS. Typically, sample blocks still show some pinkness in color. Place the samples close to the fluorescent light in the chemical fume hood, at RT O/N or longer until they appear completely clear.

3. Postembedding EM Immunogold Immunocytochemistry

1. Cut 1 µm sections with ultramicrotome, stain sections with 1% toluidine-blue, and examine them for section orientation; orient the tissue block to achieve the optimal transverse plane of sectioning.
2. Cut 70 nm thick ultrathin sections with ultramicrotome and collect them on Formvar-carbon-coated nickel slot grids.
3. Wash grids one time in distilled H₂O followed by a Tris-buffered saline three times (TBS, 0.05 M Tris buffer, 0.7% NaCl, pH 7.6) wash.
4. Incubate grids in 20 µl drops of 5% bovine serum albumin (BSA) in TBS for 30 min, and then in 20 µl drops of a mixture containing goat anti-cholera toxin subunit B (anti-CTB, 1:3,000) and an antibody to one N-methyl-D-aspartate receptor (NMDAR) subunit (anti-rabbit glutamatergic NMDAR subunit A [GluN2A] 1:50, GluN2B 1:30), or an anti-rabbit GluA2/3 (heterotetrametic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid [AMPA] receptor, 1:30) in TBS-Triton (TBST, 0.01% Triton X-100, pH 7.6) with 2% BSA and 0.02 M NaN₃ O/N at RT.
5. Wash grids on three separate drops (20 µl) of TBS (pH 7.6) for 10, 10, and 20 min, followed by TBS (pH 8.2) for 5 min.
6. Incubate grids for 2 hr on drops (20 µl) of a mixture containing donkey anti-rabbit immunoglobulin G (IgG, 1:20) coupled to 10 nm gold particles and donkey anti-goat IgG (1:20) coupled to 18 nm gold particles in TBST (pH 8.2) with 2% BSA and 0.02 M NaN₃.
7. Wash grids in 20 µl drops of TBS (pH 7.6) for 5, 5, and 10 min, then in ultrapure water and then dry them.
8. Counterstain grids with 5% uranyl acetate and 0.3% lead citrate in distilled H₂O for 8 and 5 min under dark conditions, respectively.

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
Paraformaldehyde	EMS	15710
Glutarldehyde	EMS	16019
NaH2PO4	Sigma	S9638
Na2HPO4	Sigma	7782-85-6
CaCl2	Sigma	C-8106
BSA	Sigma	A-7030
Triton X-100	Sigma	T-8787
NaOH	Sigma	221465
NaN3	JT Baker	V015-05
Glycerol	Gibco BRL	15514-011
Lowicryl HM 20	Polysciences	15924-1
Tris-Base	Fisher	BP151-500
Tris	Fisher	04997-100
Anti-GluN2A	Millipore	AB1555P	Dilution 1/50
Anti-GluN2B	Millipore	AB1557P	Dilution 1/30
Anti-GluA2/3	Millipore	AB1506	Dilution 1/30
Anti-PSD-95	Millipore	MA1–046	Dilution 1/100
Donkey anti-rabbit IgG-10 nm gold particles	EMS	25704	Dilution 1/20
Donkey anti-mouse IgG-10 nm gold particles	EMS	25814	Dilution 1/20
Donkey anti-mouse IgG-5 nm gold particles	EMS	25812	Dilution 1/20
Donkey anti-goat IgG-18 nm gold particles	Jackson ImmunoResearch	705-215-147	Dilution 1/20
Formvar-Carbon coated nickel-slot grids.	EMS	FCF2010-Ni
Uranyl acetate	EMS	22400-1
Methanol	EMS	67-56-1
Lead citrate	Leica
Leica EM AFS	Leica
Leica EM CPC	Leica
Ultromicrotome	Leica
JEOL 1200 EM	JEOL
liquid nitrogen	Roberts Oxygen
Propane	Roberts Oxygen
CTB	List Biological Laboratories	104	1-1.2%
Anti-CTB	List Biological Laboratories	703	Dilution 1/4000