TITLE: Retrograde labeling of brown adipose tissue (BAT)-projecting sympathetic neurons with cholera toxin B (CTB)
AUTHORS: Seoeun Lee, Lori Zeltser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to retrogradely label the Brown Adipose Tissue (BAT) - projecting sympathetic neruons in stellate ganglia (SG) using Cholera toxin subunit B (CTB) conjugated with Alexa Flour 555.</div></div>

[STEPS]
?. [Survival surgery for CTB injection into BAT]
Anesthetize the animal with isoflurane, and keep it anesthetized for the whole procedure
?. [Survival surgery for CTB injection into BAT]
Once animal is unconscious (check with toe pinch), place it on heating pad and lubricate the eyes with ophthalamic ointment
?. [Survival surgery for CTB injection into BAT]
Inject buprenorphine (100 µl/ 10 µg) on the surgical site for analgesia
?. [Survival surgery for CTB injection into BAT]
Remove the hair of interscapular region
?. [Survival surgery for CTB injection into BAT]
Scrub the surgical area using povidone-iodine and 70% isopropyl alcohol, 3 times each
?. [Survival surgery for CTB injection into BAT]
Open the sterilized pouch with tools, put the suture on the sterile space (save the plastic cover for CTB drops), uncover the sterile draping, place on the sterile space
?. [Survival surgery for CTB injection into BAT]
Prepare CTB in syringe (10 ul for each depot), put on sterile gloves*Alternatively, 1-2 ul of less volume can be injected using Hamilton syringe or mouth pipette with a pulled glass needle
?. [Survival surgery for CTB injection into BAT]
Cut the sterile draping to make a hole that has a size of the operating site
?. [Survival surgery for CTB injection into BAT]
Make a shallow incision at the center of the shaved area (1 cm below the head) and extend about 0.5 cm bilaterally
?. [Survival surgery for CTB injection into BAT]
Grasp the adipose tissue of one side, locate BAT inside and inject the CTB solution
?. [Survival surgery for CTB injection into BAT]
Repeat the same injection on the opposite side to complete the bilateral injection
?. [Survival surgery for CTB injection into BAT]
Gently put back the fat pad and close the wound with a 4-0 PS-4 suture
?. [Survival surgery for CTB injection into BAT]
Wipe the closed surgical wound with Povidone-iodine and alcohol prep
?. [Survival surgery for CTB injection into BAT]
Inject buprenorphine or appropriate analgesics every 4-12 hours after thesurgery for 48 hours
?. [Stellate Ganglia collection]
Sacrifice the animals 3 days after the injection, dissect out stellate ganglia (SG) on a slide with cold PBS
?. [CTB preparation ]
Dilute CTB in saline to make 1% stock solution: 500 ug CTB powder in 50 ul saline
?. [CTB preparation ]
Aliquot 4 ul of 1% CTB solution to each bottle and store it at -20ºC
?. [CTB preparation ]
When ready to use, take out the frozen CTB solution and adjust with more saline to final volume desired (ex. add 16 ul of saline each bottle to get 20 ul of total solution, which will be injected 10 ul to each depot)
?. [Stellate Ganglia collection]
Fix the dissected SG in 4% PFA in 0.1M PB for 1 hour and wash with PBS overnight at 4ºC
?. [Stellate Ganglia collection]
Attach an iSpacer on a slide glass, carefully locate the SG inside the spacer (best to stretch SG horizontally for imaging) and mount with Vectashield (~110 ul) and a cover slip
?. [Imaging]
SG was imaged with Zeiss confocal 710 using tile scan and z-stack options.