TITLE: Optogenetic Stimulation of superior mesenteric ganglion in a model of septic shock
AUTHORS: Kaitlin Murray, Jessica Sladek, Colin Reardon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol details how to use optogenetics to activate neurons in the superior mesenteric ganglion of a mouse. </div></div>

[STEPS]
?. [Optogenetic Stimulation]
Place anesthetized mouse supine on heating pad under stereo dissecting scope
?. [Optogenetic Stimulation]
Make a 1-inch incision on the right side of the mouse, directly under the rib cage
?. [Optogenetic Stimulation]
Locate the SMG
?. [Optogenetic Stimulation]
Begin optogenetc stimulation (250mA, 10Hz, 2ms) of superior mesenteric ganglion (SMG) by placing the fiber 1-2mm over SMG
?. [Optogenetic Stimulation]
Stimulate for 10 minutes
?. [Optogenetic Stimulation]
Retro-orbital IV injection of 4mg/kg LPS
?. [Optogenetic Stimulation]
Continue optogenetic stimulation of SMG for another 10 minutes
?. [Optogenetic Stimulation]
Keep tissues moist with continual application of sterile 1X PBS and cover wound with sterile gauze
?. [Optogenetic Stimulation]
Allow mouse to rest (under anesthesia) for 50 minutes
?. [Blood Collection]
Perform a cardiac puncture to draw blood serum and collect in serum separator tube
?. [Blood Collection]
Euthanize mouse
?. [Blood Collection]
Centrifuge blood at 15000rpm for 5 mins, 4C
?. [Blood Collection]
Remove top layer and store at -80C
?. [ELISA Protocol]
Coat Corning Costar 9018 ELISA plate with 100ul/Well of capture antibody bufferAdd 48ul capture antibody to 12ml 1x Coating BufferSeal plate, incubate overnight 2-8C
?. [ELISA Protocol]
Aspirate wells and wash 3x with >250ul/well washing bufferwash buffer: 1x PBS, 0.05% Tween 20Allow ~1 minute soak between washingBlot on absorbent paper
?. [ELISA Protocol]
Dilute 5x ELISA/ELISAPOT diluent with 4 parts DI water and block wells with 200ul/well of 1x ELISA/ELISAPOT diluent10ml of 5x ELISA/ELISAPOT to 40ml DI waterIncubate RT for 1 hour
?. [ELISA Protocol]
Optional: aspirate and wash with wash buffer
?. [ELISA Protocol]
Reconstitute lypohilized standards, sit for 15 minutes with gentle agitation prior to diluting further
?. [ELISA Protocol]
Dilute reconstituted standard with 1x ELISA/ELISAPOT diluentAdd 100ul of standard to 1400ul of 1x ELISA/ELISAPOT diluentAdd 100ul/well of top standard concentration to appropriate wellsPerform 2-fold serial dilutions of the top standards to make a standard curve of a total of 8 points: Add 100ul/wellInclude at least 2 wells with 100ul/well of 1x ELISA/ELISAPOT diluent as blanksSeal plate and incubate at RT for 2 hours (or overnight 2-8C)
?. [ELISA Protocol]
Aspirate and wash for 3-5 washes
?. [ELISA Protocol]
Add 100ul/well of detection antibody diluted in 1x ELISA/ELISAPOT diluent48ul detection antibody to 12ml of 1x ELISA/ELISAPOT diluentseal plate and incubate 1 hour at RT
?. [ELISA Protocol]
Aspirate and wash for 3-5 washes
?. [ELISA Protocol]
Add 100ul/well of Avidin-HRP diluted in ELISA/ELISAPOT diluent48ul enzyme to 12ml 1x ELISA/ELISAPOT diluentseal and incubate 30 minutes at RT
?. [ELISA Protocol]
Aspirate and wash, soak wells in wash buffer 1-2 minutes prior to aspiration
?. [ELISA Protocol]
Add 100ul/well of 1x TMB solution to each well
?. [ELISA Protocol]
Add 50ul of stop solution to each well
?. [ELISA Protocol]
Read plate at 450nm- if  λ subtraction is available, subtract the values of 570nm from those of 450nm and analyze