TITLE: Extraction of brain tissue with fluorogold-labelled neurons for transmission electron-microscopy imaging
AUTHORS: enrico.zampese

[DESCRIPTION]
Protocol for injection of Fluorogold particles for retrograde labelling or neurons, perfusion, fixation, and tissue extraction for Transmission Electron Microscopy observation.

This protocol is intended for the preparation of samples to be processed and imaged at the Northwestern University Center for Advanced Microscopy (NU CAM; https://www.feinberg.northwestern.edu/sites/cam/index.html).




[GUIDELINES]
This protocol is intended for the preparation of samples to be processed and imaged at the Northwestern University Center for Advanced Microscopy (NU CAM; https://www.feinberg.northwestern.edu/sites/cam/index.html).

[STEPS]
SECTION: Stereotactic injection of fluorogold particles in the brain
1. Prepare the fluorogold working solution by dissolving fluorogold powder in the appropriate volume of sterile NaCl 0.9% saline. Recommended working concentration: 1% fluorogold

SECTION: Stereotactic injection of fluorogold particles in the brain
2. Prepare a clean empty mouse cage on a heating pad and a clean mouse cage with gel food for post-operatory care.
SECTION: Stereotactic injection of fluorogold particles in the brain
3. Set up sterile working area including stereotaxic frame
SECTION: Stereotactic injection of fluorogold particles in the brain
4. Measure the weight of the mouse
SECTION: Stereotactic injection of fluorogold particles in the brain
5. Anesthetize mouse in induction chamber (recommended: 2.5% isoflurane, 200ml/min flow rate)
SECTION: Stereotactic injection of fluorogold particles in the brain
6. Hair over surgery area can be quickly clipped before transferring the mouse onto the stereotaxic frame.
SECTION: Stereotactic injection of fluorogold particles in the brain
7. Once the mouse is deeply anesthetized (~5 min), stop anesthesia and move the mouse to the stereotaxic frame over the heating pad with the temperature probe and secure the mouse mouth on the nose cone. Restart anesthesia (directed towards the nose cone).
SECTION: Stereotactic injection of fluorogold particles in the brain
8. The heating pad settings should be adjusted so that the temperature probe placed under the mouse should read a body temperature between 33-37C.
SECTION: Stereotactic injection of fluorogold particles in the brain
9. Apply ophthalmic ointment over eyes
SECTION: Stereotactic injection of fluorogold particles in the brain
10. Inject appropriate volume (based on mouse weight and recommended dosage) of analgesic; an appropriate amount of saline can also be injected to prevent dehydration during the procedure.
SECTION: Stereotactic injection of fluorogold particles in the brain
11. Carefully insert and secure the ear-bars. The position of the mouse head will be verified and adjusted once the skull is exposed, but it is recommended to make sure that the head is not visibly tilted.
SECTION: Stereotactic injection of fluorogold particles in the brain
12. Clean the area of the incision with the povidone-iodine swab followed by the ethanol swab. Repeat 3 times.
SECTION: Stereotactic injection of fluorogold particles in the brain
13. It is preferred to apply line-block anesthetic (0.15% bupivacaine) under the skull skin before starting the procedure rather than applying EMLA cream on the sutured skin at the end of the surgery.
SECTION: Stereotactic injection of fluorogold particles in the brain
14. With the fine scissor, expose the skull by making an anterior-posterior incision
SECTION: Stereotactic injection of fluorogold particles in the brain
15. Visually identify bregma and lambda.
SECTION: Stereotactic injection of fluorogold particles in the brain
16. Insert a glass pipette (a small volume of non-toxic food dye can be used to help marking the relevant spots) on the stereotaxic arm holder and lower it onto the skull.
SECTION: Stereotactic injection of fluorogold particles in the brain
17. Mark bregma by gently touching the intersection of the coronal/sagittal sutures with the pipette tip, and zero the coordinates on the reader.
SECTION: Stereotactic injection of fluorogold particles in the brain
18. Move to lambda (intersection of lambdoid and sagittal sutures) and measure its position relative to bregma.
SECTION: Stereotactic injection of fluorogold particles in the brain
19. Minimize the deviation of dorso/ventral (D/V) and medio/lateral (M/L) distance between lambda and bregma by adjusting the position of the head.
SECTION: Stereotactic injection of fluorogold particles in the brain
20. Zero the coordinates at bregma and repeat bregma/lambda measurements until satisfactory.
SECTION: Stereotactic injection of fluorogold particles in the brain
21. Once the head is in the correct position, it is possible to identify the desired injection spot.
SECTION: Stereotactic injection of fluorogold particles in the brain
22. It is recommended to use the measured anterior/posterior (A/P) distance between bregma and lambda to calculate an adjustment factor for the final coordinates: the measured B-L distance will be divided by the reference distance of 4.21. For an adult mouse, the obtained value (“adjustment ratio”) should be close to 1, and in this case no coordinates adjustment is required (but still optional). For smaller mice, the reference coordinates should be multiplied by the calculated adjustment ratio to obtain the final coordinates for the specific mouse.
SECTION: Stereotactic injection of fluorogold particles in the brain
23. Move the pipette to the spot indicated by the adjusted A/P and M/L coordinates and mark it.
SECTION: Stereotactic injection of fluorogold particles in the brain
24. Whether performing uni-lateral or bi-lateral injections, it is recommended to mark the spots on both sides of the skull, and to measure their relative dorso-ventral position. Their relative D/V deviation of the right and left spots should be minimized by adjusting the position of the head.
SECTION: Stereotactic injection of fluorogold particles in the brain
25. Once the desired spot has been marked, the marker pipette can be removed, and a hole is drilled in the skull at the indicated position.
SECTION: Stereotactic injection of fluorogold particles in the brain
26. Blood and debris are cleaned with sterile saline and sterile cotton swabs.
SECTION: Stereotactic injection of fluorogold particles in the brain
27. Insert micropipette with volumetric references in the holder and connect it to a syringe to apply positive/negative pressure.
SECTION: Stereotactic injection of fluorogold particles in the brain
28. Draw up desired volume of fluorogold solution in the syringe by applying negative pressure.
SECTION: Stereotactic injection of fluorogold particles in the brain
29. Lower pipette loaded with the fluorogold solution into the hole until the tip touches the dura. Zero the dorso-ventral coordinate.
SECTION: Stereotactic injection of fluorogold particles in the brain
30. Gradually lower the pipette tip into the brain until the desired dorso-ventral coordinate is reached.
SECTION: Stereotactic injection of fluorogold particles in the brain
31. Slowly inject the desired volume of fluorogold solution (recommended: ~150nl/min) by gently and gradually applying positive pressure.
SECTION: Stereotactic injection of fluorogold particles in the brain
32. Release pressure and leave the pipette in position for ~5-10min so that the fluorogold solution can spread and be absorbed by the tissue.
SECTION: Stereotactic injection of fluorogold particles in the brain
33. Slowly retract injection pipette.
SECTION: Stereotactic injection of fluorogold particles in the brain
34. Suture skin.
SECTION: Stereotactic injection of fluorogold particles in the brain
35. Optional: repeat saline injection to prevent dehydration
SECTION: Post-surgery care
36. Remove animal from stereotaxic frame and place it in the clean, empty cage on heating pad until deambulatory (~10-15min).
SECTION: Post-surgery care
37. Once awake and deambulatory, mouse can be moved to the clean cage with gel food, also on heating pad.
SECTION: Post-surgery care
38. 24 hours after surgery, a second dose of Metacam is administered and antibiotic ointment is applied on the sutured skin.
SECTION: Post-surgery care
39. The health status of the mouse is monitored over the following days. If needed, additional doses of Metacam or saline can be administered.
SECTION: Post-surgery care
40. Mouse is normally kept in a cage on heating pad for at least 4 days and is then moved to standard housing.
SECTION: Post-surgery care
41. Mice are sacrificed for tissue extraction 5-6 days after fluorogold injection.
SECTION: Perfusion and fixation
42. Under the hood, pour PBS and fixative solutionsin 50ml tubes.
SECTION: Perfusion and fixation
43. Attach the perfusion needle to the connector atthe end of the tubing
SECTION: Perfusion and fixation
44. Start running PBS through the tubing.
SECTION: Perfusion and fixation
45. If possible, it is recommended to have aconnector system with a switch that allows to pre-load the fixative solutionand the PBS solution in the respective collection tubing and quickly switchfrom one to the other avoiding the need to move a single collection tube fromone solution to the other, interrupting the procedure.
SECTION: Perfusion and fixation
46. A tubing connected to a vacuum-trap can be usedto immediately collect waste solution during the procedure.
SECTION: Perfusion and fixation
47. Terminally anesthetize the mouse according to institutional protocols.
SECTION: Perfusion and fixation
48. Bring the anesthetized mouse to the dissection tray and verify that the mouse is fully anesthetized. This can be performed by pinching one of the posterior paws and observing the presence (or lack of) pain reflex. The mouse must be fully anesthetized before starting the trans-cardiac perfusion.
SECTION: Perfusion and fixation
49. Once full anesthesia is achieved, the mouse can be positioned and secured on the dissection tray.
SECTION: Perfusion and fixation
50. The mouse should be positioned in a supine position, with the head oriented away from the operator. If possible, the dissection tray should be slightly inclined, with the mouse oriented so that the head is slightly pointing down-ward.
SECTION: Perfusion and fixation
51. Holding the skin just below the sternum with a tweezer, cut the skin just below, exposing the peritoneal cavity and the rib cage. The diaphragm should remain intact.
SECTION: Perfusion and fixation
52. Expand the cut and with the scissors cut the fascia connecting the skin to the rib cage, toward the head.
SECTION: Perfusion and fixation
53. Once the rib cage is clearly visible, carefully cut the diaphragm without damaging the beating heart. Cut the rib cage and lift it toward the head. A needle can be used to hold it in position while operating. The liver should be visible in the abdominal cavity.
SECTION: Perfusion and fixation
54. Carefully insert the needle connected to the peristaltic pump (where PBS is circulating) in the left ventricle of the heart, and rapidly pinch the right atrium with the spring scissor. Dark-red blood should start flowing out of it immediately. Hold the needle in position, while the solution washes out the blood from the mouse. The heart should still be beating. A wash water bottle can be used to remove excess blood and see more clearly.
SECTION: Perfusion and fixation
55. As soon as the liver start to look depleted of blood, shift the perfusing solution to fixative.
SECTION: Perfusion and fixation
56. Maintain the needle in position and rinse with water.
SECTION: Perfusion and fixation
57. In general, we recommend perfusing each mouse with a volume of fixative approximately corresponding to their weight in grams (e.g.: 20 ml of fixative solution for a 20g mouse).
SECTION: Perfusion and fixation
58. As the fixative reaches the tissues, some appendages of the mouse might start to move or contract. This normally indicates that the fixation is working. If the perfusion is done correctly, this should start shortly after changing the perfusion solution to fixative.
SECTION: Perfusion and fixation
59. Once the desired volume of fixative has run through, stop the perfusion, remove the needle and release the mouse. The carcass should be very stiff.
SECTION: Perfusion and fixation
60. Decapitate the mouse with the scissor.
SECTION: Perfusion and fixation
61. With the fine scissors, cut the skin and expose the median line of the skull. Cut off the posterior part of the skull. Then, carefully cut along the median line, towards the rostral part of the head. Past bregma, apply two diagonal cuts toward the eyes, and two other later cuts along the lambdoidal sutures.
SECTION: Perfusion and fixation
62. Carefully open the skull with the help of the tweezers and expose the brain.
SECTION: Perfusion and fixation
63. With the spatula, gently remove the brain and slide it into the remaining fixative solution in the falcon tube. Close the falcon tube and gently shake it.     
SECTION: Perfusion and fixation
64. Properly dispose of the mouse carcass in the carcass bag.
SECTION: Perfusion and fixation
65. Dispose of all the fixative waste (liquid and solid) and sharps according to institutional guidelines.
SECTION: Perfusion and fixation
66. Clean/wash all the tools/equipment.
SECTION: Perfusion and fixation
67. Leave the brain in fixative overnight at 4C.
SECTION: Tissue extraction and isolation
68. Shortly before the slices collection, remove fixative solution where the brain has been kept overnight and rinse 3 times for 10 minutes each with PBS. Keep the brain in a clean tube with fresh PBS.  
SECTION: Tissue extraction and isolation
69. Cut a 0.5x0.5in rectangle of agarose
SECTION: Tissue extraction and isolation
70. The recommended settings for slicing are: 400um thickness, 1mm oscillation amplitude, speed ~0.2-0.3mm/s
SECTION: Tissue extraction and isolation
71. Obtain fixed thick slices:
SECTION: Tissue extraction and isolation
71.1. Remove the fixed brain from PBS and place it on the weighing bowl/petri dish.
SECTION: Tissue extraction and isolation
71.2. With the single edge blade, cut the brain according to the desired slicing orientation. For coronal midbrain slices, it is recommended to cut off the more rostral part of the brain, creating a flat surface that will be used to glue the brain to the slicing chamber stage, and to remove the cerebellum.
SECTION: Tissue extraction and isolation
71.3. Apply super glue to the slicing chamber stage.
SECTION: Tissue extraction and isolation
71.4. Gently glue the brain onto the stage, in the desired position. For coronal midbrain slices, it is recommended to position the ventral part of the brain facing the blade, with the rostral side at the bottom. If desired, glue also the piece of agarose on the side of the brain that will not be facing the blade.
SECTION: Tissue extraction and isolation
71.5. Place the cutting stage in the chamber, set the chamber in the vibratome tray and attach it to the vibratome.
SECTION: Tissue extraction and isolation
71.6. Fill the cutting chamber with PBS.
SECTION: Tissue extraction and isolation
71.7. Lower the blade holder (with blade already inserted) into the chamber and adjust the position/settings of the blade.
SECTION: Tissue extraction and isolation
71.8. Start cutting.
SECTION: Tissue extraction and isolation
71.9. While still far from the region of interest, it is possible to manually move the blade closer to the target area rather than cutting thin slices throughout the entire tissue, unless desired.
SECTION: Tissue extraction and isolation
71.10. Discard debris/excess tissue.
SECTION: Tissue extraction and isolation
71.11. When at the target area, cut 400-500um slices, gently collect them with a transfer pipette or a tweezer and move them to one of the wells of the collection plate. It is recommended to keep track of the collection order for anatomical accuracy. It is also recommended to keep each slice in a separate well in a plate, so that it will be easier to identify the one with the best fluorogold signal.
SECTION: Tissue extraction and isolation
71.12. Once all the slices are collected, stop the vibratome, return the blade holder to a safe position, discard extra solution and what remains of the brain tissue accordingly.
SECTION: Tissue extraction and isolation
73. Carefully dispose of all the waste and sharps according to institutional guidelines.
Wash the apparatus and the tools and clean the working station.
SECTION: Tissue extraction and isolation
72. Isolation of region containing Fluorogold-labelled cell
SECTION: Tissue extraction and isolation
72.1. Move the plate with the slices to the microscope station.
SECTION: Tissue extraction and isolation
72.2. Turn on the microscope and observe slices with the appropriate fluorogold-compatible filter set.
SECTION: Tissue extraction and isolation
72.3. Identify the slice with the best fluorogold signal and sketch on some paper the exact anatomical location of the area of interest. Accurately tracking the position of the fluorogold labelled cells will be very important for the following steps.
SECTION: Tissue extraction and isolation
72.4. Move the selected slice to a petri dish with some PBS and using the tweezers and the disposable scalpel dissect a 1mm by 1mm square of tissue containing the area of interest. It is recommended to carefully sketch the exact distribution of the fluorogold-labelled area also within the tissue square.
SECTION: Tissue extraction and isolation
72.5. Under a chemical hood, transfer the selected block of tissue to the hinged plastic vial containing fixative solution.
SECTION: Tissue extraction and isolation
72.6. The tissue block can be stored in the vial with fixative at 4C.
SECTION: Tissue extraction and isolation
74. This protocol is intended for the preparation of samples to be processed and imaged at the Northwestern University Center for Advanced Microscopy (NU CAM) The vial containing the selected tissue will be transferred to the Electron Microscopy core for further processing. Provide copies of the sketches identifying the exact position of the region of interest to the Core personnel.
SECTION: Tissue extraction and isolation
75. Electron Microscopy core will proceed with processing the tissue for electron microscopy observation according to their protocol (https://www.feinberg.northwestern.edu/sites/cam/docs/protocols/tem-methods.pdf).
SECTION: Tissue extraction and isolation
75.1. This includes post-fixation in buffered 2% OsO4, staining in 3% uranyl acetate; dehydration in ascending ethanol; transition to propylene oxide and embedding in Embed 812 (Electron Microscopy Sciences). 
SECTION: Tissue extraction and isolation
75.2. The sample will be then sectioned using an ultramicrotome (Leica Ultracut UC6). 1um thick sections are stained with Toluidine Blue O and 70 nm sections are collected on 200 mesh copper grids and stained with uranyl acetate and Reynolds lead citrate.
SECTION: Tissue extraction and isolation
75.3. The 70nm are now ready for electron microscopy imaging.
SECTION: Transmission Electron Microscopy
76. The operation of the transmission electron microscopy station requires prior training, observation, and approval by the NU CAM Core personnel. Please refer to the training module/instructions for all the operations.

SECTION: Transmission Electron Microscopy
77. Image acquisition
SECTION: Transmission Electron Microscopy
77.1. Full microscope apparatus is turned on an operated according to specific instructions
SECTION: Transmission Electron Microscopy
77.2. Liquid nitrogen is used as a cooling agent, as instructed (handle with cryo-gloves)
SECTION: Transmission Electron Microscopy
77.3. Carefully insert one sample grid in the microscope apparatus according to instructions.
SECTION: Transmission Electron Microscopy
77.4. The transmission electron microscope is operated at 80kHz

SECTION: Transmission Electron Microscopy
77.5. Different levels of magnification can be adopted.
SECTION: Transmission Electron Microscopy
77.6. At lower magnification, identify cell bodies with morphological features corresponding to the cells of interests.
SECTION: Transmission Electron Microscopy
77.7. Identify retrogradely-labelled cells by the presence of cytological structures that indicate Fluorogold uptake (e.g.electron-dense vesicular bodies, multilamellar electron-dense vesicles).
SECTION: Transmission Electron Microscopy
77.8. Collect high magnification/high resolution images.
SECTION: Transmission Electron Microscopy
77.9. Export and save the collected images.
SECTION: Transmission Electron Microscopy
78. Carefully shut off microscope station according to instructions.
SECTION: Transmission Electron Microscopy
78.2. Collect residual liquid nitrogen (handle with cryo-gloves).

SECTION: Transmission Electron Microscopy
78.1. Carefully remove grid from microscopy apparatus according to instructions.
SECTION: Transmission Electron Microscopy
78.3. Carefully clean the work station.