Dissect tissues .
{"action": "dissect", "output": "", "reagent": ["tissues"]}
postfix them in 4% PFA for 4 h at room temperature
{"action": "patch", "output": "", "reagent": ["4% PFA"], "time": ["4 h"], "temperature": ["room temperature"]}
Dehydrate tissue samples in 30% sucrose solution.
{"action": "dehydrate", "output": "", "reagent": ["30% sucrose solution"]}
Prepare thin tissue sections on a Cryostat microtome.
{"action": "prepare", "output": "", "device": ["Cryostat microtome"], "container": ["thin tissue sections"]}
Wash sections in 0.3% Triton X-100 in PBS (PBST) block them in 2% BSA in PBST at room temperature for 1 h. 2. #####%%%%% output "samples" are used in line12
{"action": "wash", "output": "samples", "reagent": ["0.3% Triton X-100 in PBS (PBST)"]}
Wash samples three times in PBST. #####%%%%% output "HCR amplification buffer" are used in line14
{"action": "wash", "output": "HCR amplification buffer", "reagent": ["samples"]}
incubate samples in HCR amplification buffer #####%%%%% output "secondary antibodies" are used in line16
{"action": "incubate", "output": "secondary antibodies", "reagent": ["HCR amplification buffer"], "temperature": [""], "time": [""]}
Incubate sections with biotinylated secondary antibodies conjugated secondary antibodies. #####%%%%% output "samples" are used in line18
{"action": "detect", "output": "samples", "reagent": ["secondary antibodies"]}
Wash samples three times in PBST.
{"action": "wash", "output": "", "reagent": ["samples"]}
Both amplifiers are then added to amplification buffer to a final concentration of 12.5 nM. #####%%%%% output "GO" are used in line24
{"action": "add", "output": "GO", "container": ["amplification buffer"], "concentration": ["12.5 nM"], "reagent": [""]}
Optional: Graphene oxide (GO) can be added for applications that demand background suppression.

Add GO to the buffer from step 2 to 20 µg/mL thoroughly. #####%%%%% output "samples" are used in line26
{"action": "add", "output": "samples", "reagent": ["GO"], "concentration": ["20 µg/mL"]}
Wash samples three times in PBST.
{"action": "wash", "output": "", "reagent": ["samples"]}