1.Using a cryostat, cut the lumbar segments where the double-infected cell bodies reside
{"action": "cut", "output": "", "device": ["a cryostat"]}
Cut the tissue at 30 µm in sets of 5, mounting the sequential cross-sections across each slide until there are five cross-sections per slide

first section cut mounted to slide A1, second section – B1, .
{"action": "display", "output": "", "time": [""], "string": [""]}
Store slides at -20ºC.
{"action": "rotate", "output": "", "temperature": ["-20\u00baC"]}
Once at temperature, remove a complete set of slides from -20ºC . #####%%%%% output "the blocking solution" are used in line16
{"action": "remove", "output": "the blocking solution", "reagent": [""]}
outline the sections using a hyprophobic pen

4.During the hydration step, prepare the blocking solution \0.1-0.3% Triton x-100
{"action": "prepare", "output": "", "reagent": ["the blocking solution"]}
5.Apply the blocking solution, let incubate for 1 hour. #####%%%%% output "1:5,000 rabbit anti-GFP (abcam ab290) -NeuN (Millipore ABN90P)" are used in line18
{"action": "incubate", "output": "1:5,000 rabbit anti-GFP (abcam ab290) -NeuN (Millipore ABN90P)", "reagent": ["the blocking solution"], "time": ["1 hour"], "temperature": [""]}
Make a cocktail solution with the following primary antibodies: 1:5,000 rabbit anti-GFP \(abcam ab290) -NeuN \(Millipore ABN90P).
{"action": "make", "output": "", "reagent": ["1:5,000 rabbit anti-GFP (abcam ab290) -NeuN (Millipore ABN90P)"]}
9.If detecting synaptic co-localization of GFP-enhanced viral eTeNT.EGFP, perform the following steps: \(a) rinse the slides in 0.1 M TBS three times, . #####%%%%% output "anti-mouse AlexaFluor647" are used in line22
{"action": "perform", "output": "anti-mouse AlexaFluor647", "frequency": ["three times"], "reagent": [""]}
If staining is for synaptic co-localization, include anti-mouse AlexaFluor647 at 1:200 \(JacksonImmunoresearch 715-605-151). #####%%%%% output "synaptic co-localization" are not used
{"action": "include", "output": "synaptic co-localization", "reagent": ["anti-mouse AlexaFluor647"]}
During the second wash, add hoechest \(1:1,000) to stain for nuclei.
{"action": "add", "output": "", "reagent": ["hoechest (1:1,000)"]}