TITLE: Virus injection
AUTHORS: Liu Liu, Susu Chen, Nuo Li, Karel Svoboda

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to inject viral vectors expressing proteins such as ion channels, transcription factors, enzymes, receptors, fluorescent proteins or other non-toxic, non-hazardous payloads. The viral vectors include AAV, Adenovirus, modified RV, Lentivirus, Modified Herpes Simplex Virus, and Pseudorabies Virus.</div><div class = "text-block">The protocol is often performed in conjunction with the headbar implantation protocol (dx.doi.org/10.17504/protocols.io.bcrsiv6e). Please refer to the headbar implantation protocol for more details.</div></div>

[STEPS]
?. [Pipette preparation]
Using thin-walled Drummond glass, pull symmetrically on the P-2000 (Sutter) or equivalent.
?. [Pipette preparation]
Under visual inspection (e.g. microforge or microscope) inspect the tip and carefully break the tip by hitting it against a solid surface. Aim for approximately 20μm tip diameter.
?. [Pipette preparation]
Mount pipette on a beveler at a 35° angle or less to horizontal, with the broken end roughly flush with the platter. Wet a kimwipe with 70% EtOH. Start the hard drive. Maintain a wet drive surface by holding the wet wipe softly above the platter. The EtOH prevents glass dust from kicking up into the pipette. Lower the pipette and very briefly touch the spinning surface and return.
EtOH going inside through the tip of the pipette during sharpening usually indicates a successfully sharpened tip.
?. [Pipette preparation]
Inspect the pipette. Try to blow dust out of the inside and off the tip with a canned air.
If you did a bad job it looks dusty like this.  If you did a good job it looks clean and sharp like this.
?. [Pipette preparation]
Bevel at least 3 pipettes and arrange by quality. Use the best one first, with the others as backup (if you break / clog the first).
?. [Injection setup]
Thaw virus aliquot (2μL) on ice.
?. [Injection setup]
Sterilize the workspace and prepare tools. See headbar protocol for more details.
?. [Injection setup]
Cut gel foam into 1mm cubes and soak in cortex buffer
?. [Injection setup]
Adjust the alignment of the pipette holder and the plunger holder. The two attachment channels should be straight and co-linear.
?. [Injection setup]
Place a tiny platform adjacent to the mouse head holder and wrap with parafilm for virus loading.
?. [Injection setup]
Fill the pipette with mineral oil, avoiding bubbles.
?. [Injection setup]
Mount the injector, eject any bubbles, then carefully suck up the virus from a drop on the parafilm.
?. [Surgery and injection]
Wet the skull with cortex buffer to visualize the vasculature.
?. [Surgery and injection]
Confirm the target, dry the skull and drill a small hole directly above the target. Look carefully for cracking or peeling of the bone, this indicates you are at or very near the dura, and can stop drilling.
?. [Surgery and injection]
Re-wet the area and zoom in.
?. [Surgery and injection]
Position the pipette directly above the craniotomy, Watch out to not hit the hydraulics on the injector with the scope support.
?. [Surgery and injection]
Eject a small volume of virus to remove any bubbles and wick away the drop using a saline soaked piece of gel foam. Keep the lighting at a fair distance from the pipette, at moderate brightness, to reduce volume expansion from heating. Once the lighting is set, do not change it so that the pipette stays in a thermal steady state.
?. [Surgery and injection]
Position the needle just onto the surface of the craniotomy. Zero the manipulator.
?. [Surgery and injection]
Lower the pipette to the desired depth at 2 μm/s, and inject the desired amount (typically 10-100 nL) of virus at 0.5 nL/s.
?. [Surgery and injection]
Test the pipette for clogs by ejecting a small volume.
If it comes out easily, the injection probably worked. If nothing comes out, don't despair, it might have gotten clogged on the way out. If clogged, then repeat the injection with a new pipette.
?. [Surgery and injection]
Wipe the pipette with wet gel foam, then eject another tiny drop to seal the end. If you thinned a large area of skull, coat the thinned area with a small volume of Krazy glue. Fill well with Kwik-Cast and cover with dental acrylic.
?. [Surgery and injection]
Dispose of virus handling parts in a labeled sealed 50mL conical.
?. [Surgery and injection]
After each injection, leave the pipette in tissue for  . Maintain a wet surface on the craniotomy to prevent tissue sticking to the pipette.
?. [Surgery and injection]
Slowly retract the pipette out of the brain at 5-10 μm/s. After the pipette is removed from the brain, there should be no bleeding at the injection site.
?. [Injection setup]
Mount the pipette in the pipette holder, and insert the plunger into the back end of the pipette. Turn the knob of Narishige injection system clockwise to eject and counter-clockwise to suck.