TITLE: Pseudorabies virus (PRV) injection into inguinal white adipose tissue
AUTHORS: Clara Huesing, Heike Muenzberg, Rui Zhang, Nathan Lee, Emily Qualls-Creekmore, Marie Francois

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We utilize pesudorabies virus (PRV) retrograde tracing in combination with reporter mice, iDISCO, and confocal/light sheet microscopy to identify the location of pre- and post-ganglionic neurons as well as nerves that selectively innervate the iWAT in the mouse.</div></div>

[STEPS]
?. [Room preparation]
Turn on the glass bead sterilizer
?. [Room preparation]
Turn on the two heating pads (stereotaxic platform and designated area for recovery)
?. [Room preparation]
Place a non-sterile towel on your recovery heating pad and a sterile towel on the stereotaxic platform heating pad
?. [Room preparation]
Place a sterile towel to the right of the stereotaxic platform along with your rack with the three tubes filled with ‘Novalsan’, ‘70% Isopropanol’ and ‘Empty’
?. [Room preparation]
Place multiple sterile cotton swabs into each 50mL tube
?. [Room preparation]
Put an empty cage half way on the recovery heating pad
?. [Room preparation]
Fill one 3mL syringe completely with sterile saline, place underneath your stereotaxic heating pad to warm fluid
?. [Room preparation]
Fill your 3mL syringes with sterile saline (~1.5ml for 25g mouse), place underneath your recovery heating pad to warm fluid
?. [Room preparation]
Fill your 0.5ml syringes with Bupivacaine/lidocaine (0.1ml)
?. [Room preparation]
Open the O2 tank valve, set the O2 level to 1% on the anesthesia machine
?. [Room preparation]
Check your isoflurane level on your anesthesia machine, set to 5% for the induction box
?. [Room preparation]
Place all of your surgical tools along with your eye lubricant, wound clip applicators, spare wound clips, sterile gauze and timer on your sterile towel to the right of your stereotaxic platform
?. [Virus preparation]
Place aluminum foil on clean surface, this will be the designated area to test and fill syringe with PRV
?. [Virus preparation]
Place rack with three tubes filled with ‘70% Ethanol’, ‘dH2O’, Waste
?. [Virus preparation]
Test flow of syringe by filling with dH2O and flushing several times. If you do not observe a steady flow of water out of the syringe, one must replace the syringe as this indicates the virus will not be properly administered.
?. [Pre-incision]
Record the mouse’s body weight and (if applicable) ear tag number.
?. [Pre-incision]
Place mouse into induction chamber with isoflurane set to 5%.
?. [Pre-incision]
Once the mouse is unconscious, turn your isoflurane to 1.8% (adjust this number according to the mouse’s breathing), open your stereotaxic valve, close your induction box valve and place into the stereotaxic nose cone, using a cotton swab to align its incisors into the hole.
?. [Pre-incision]
Secure your nose cone by tightening the top screw, gently tug on the base of the mouse’s tail to check if its incisors are inside the hole and the mouse is secured in the cone.
?. [Pre-incision]
Gently pull one of the mouse’s hind limbs to check its withdrawal reflex for anesthetic depth.
?. [Pre-incision]
Lubricate the mouse’s eyes
?. [Pre-incision]
Spray interscapular region with a small amount (enough to cover carprofen injection spot) 70% ethanol and inject carprofen (10ul-20ul/gram body weight) subcutaneously.
?. [Pre-incision]
Place a kimwipe underneath the mouse and shave the right dorsolumbar region of the mouse and remove all hair
?. [Pre-incision]
Disinfect freshly shaved area by lightly scrubbing skin with cotton swabs soaked in isopropanol and Novalsan. Perform three alternating scrubs of isopropanol then Novalsan.
?. [Incision]
Start your incision by pulling straight up on the skin at the midline of the back, parallel to the hip with forceps and make a transverse cut that extends the length of the dorsolumbar portion of the iWAT fat pad.
?. [Incision]
Using a sterile cotton swab, remove any connective tissue underneath your incision to spread incision apart.
?. [Incision]
Remove connective tissue underneath the skin incision using a sterile cotton swab until the iWAT depot is fully accessible.
?. [Incision]
0pt;font-family:"Times New Roman",serif'>Apply sterile saline to sterile gauze and place it over the iWAT to keep the tissue moist, then prepare the Hamilton syringe for injection.
?. [Incision]
Fill syringe with PRV-152 virus, return to the surgery platform with syringe to begin injections.
?. [Incision]
Remove the saline-soaked gauze from the iWAT
?. [Incision]
Administer injections (10x200nl) into the right iWAT pad and hold the syringe in place for ~30 seconds after each injection to minimalize backflow, slowly pull the syringe out of the iWAT while holding a sterile cotton swab on the site of injection to dry of any possible virus leakage.
?. [Incision]
Place a fresh, saline-soaked gauze on top of the iWAT and clean the syringe with 4-5 alternating flushes of 70% ethanol and dH2O. Collect flushed liquid and dispose as biohazard waste
?. [Incision]
Note: Syringe should be held parallel, not perpendicular, to the fat pad while injecting. This will help prevent piercing into the peritoneal wall, causing off-target labeling
?. [Closing incision]
Remove the saline-soaked gauze from the iWAT and with a sterile cotton swab and teethed forceps, reposition the overlaying skin back to its original location prior to incision.
?. [Closing incision]
With teethed and tissue forceps, pull up on the skin surrounding the incision in order to align both sides of incision properly, and with tissue forceps, begin going along the incision ensuring that the subcutaneous layer of skin is not sticking out past the external layer of the skin to ensure proper healing of incision.
?. [Closing incision]
Using teethed forceps and wound clip applicators, start at the side of the incision closest to the midline, and administer wound clips ~25mm apart from one another to allow for proper blood circulation for healing of the incision.
?. [Closing incision]
Once you have applied a sufficient amount of wound clips (usually 4-5), use your hemostats to tighten each of the wound clips to avoid the mouse scratching them off. Make sure not to tighten them too much or they will cause irritation and poor circulation around the wound.
?. [Closing incision]
Using teethed forceps, gently lift up skin at one end of incision and inject 0.05mL of bupivacaine/lidocaine subcutaneously. Repeat at other end of incision.
?. [Closing incision]
Turn isoflurane off, loosen up the nose cone and remove the mouse’s incisors with the wooden end of a cotton swab from the hole on the mount.
?. [Closing incision]
Scruff the mouse and turn it over to inject ~1.5mL (for a 25g mouse) of warm saline intraperitoneally
?. [Closing incision]
Place the unconscious mouse on the warm side of the cage that is half way on the recovery heating mat. Place mouse on its side.
?. [Closing incision]
Using water from animal's cage, wet a chow pellet and place on floor of cage so mouse can easily access food.
?. [Closing incision]
Place any tools used during surgery in the glass bead sterilizer for 15-20 seconds and change gloves.
?. [Post-operative Care]
1 day post-op: Administer carprofen subcutaneously (approx. 10ul/g bw), record weight of mouse, health check
?. [Post-operative Care]
2 days post-op: Administer carprofen subcutaneously (approx. 10ul/g bw), record weight of mouse, health check
?. [Post-operative Care]
3 – 7 days post-op: record weight of mouse, health check
?. [Post-operative Care]
10 days post-op: remove wound clips, replace PVC pipe in cage
?. [Pre-incision]
Wipe loose fur off mouse using kimwipe placed underneath mouse