eamag/protocols-with-steps huggingface dataset:
TITLE: Make LB agar plates to grown bacteria without antibiotic selection.
AUTHORS: Cancer Research UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Make LB agar plates to grown bacteria without antibiotic selection.</div></div>

[STEPS]
?. 
?. ABC1IngredientsQuantity2LB Agar 1L3Petri dishes90mm304 140mm10
ABC1IngredientsQuantity2LB Agar 1L3Petri dishes90mm304 140mm10
?. A1Melt LB Agar in autoclave (media melt cycle).2Allow the medium to cool to&nbsp; ~60oC in the water bath. Mix on a magnetic stirrer (to prevent air
  bubbles). Transfer to the laminar flow hood to pour the plates.3Pour plates directly from the bottle. Allow
  30-35ml/90mm dish.
    To remove bubbles from the plate flame the surface of the plate with a
  bunsen burner&nbsp; before the agar
  sets.
    4Label:&nbsp; LB
  plates have NO stripe.&nbsp;&nbsp;&nbsp;&nbsp; Invert and store @ 4oC.&nbsp;&nbsp;&nbsp; Storage life:&nbsp; ~2 week&nbsp;&nbsp;5Lab coats must be worn.
A1Melt LB Agar in autoclave (media melt cycle).2Allow the medium to cool to&nbsp; ~60oC in the water bath. Mix on a magnetic stirrer (to prevent air
  bubbles). Transfer to the laminar flow hood to pour the plates.3Pour plates directly from the bottle. Allow
  30-35ml/90mm dish.
    To remove bubbles from the plate flame the surface of the plate with a
  bunsen burner&nbsp; before the agar
  sets.
    4Label:&nbsp; LB
  plates have NO stripe.&nbsp;&nbsp;&nbsp;&nbsp; Invert and store @ 4oC.&nbsp;&nbsp;&nbsp; Storage life:&nbsp; ~2 week&nbsp;&nbsp;5Lab coats must be worn.
TITLE: Protocol 2: LAMP
AUTHORS:  

[STEPS]
?. Get FASTA sequenceYou’ll need to look up the gene for the sequence you are trying to amplify and download the FASTA file for it.
?. Go to Primer ExplorerYou will need to go to the Primer Explorer webpage, http://primerexplorer.jp/lampv5e/index.html, in order to upload your FASTA file.
?. Select which primers to choose fromOnce the the list of primers has been generated, you can now press display to see the various options for primers you can design
?. Confirm your choice. The confirmation page will compile all of the necessary primer information onto one page so that you can have one last check of the aspects of your primers before you order. When you are confident in your choice, save the primer information and send them out to be synthesized.
?. Take a screenshot of the confirmation page with all of the primer results and upload it to your lab notebook.
TITLE: UC Davis - Echocardiography
AUTHORS: Anil Singapuri

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">Cardiac hypertrophy is one of the most common causes of heart failure. The development of cardiac hypertrophy and failure can be monitored using noninvasive imaging with echocardiogram in conscious animals.</div><div class = "text-block"><span style = "font-weight:bold;">Modified from</span><span>: Timofeyev V. et al. J Mol Cell Cardiol. 2006 Jul;41(1):170-81.</span></div></div>

[STEPS]
?. The animals’ chest will be shaved and then be placed in a supine position on a warmed (37°C) towel covered surgical table.
?. The mice are restrained in a 50 ml conical tube with an opening for the nose and chest, a standard procedure for imaging mice for this kind of study.
?. The imaging generally lasts for only 2-5 min, we were trained in this method by CLAS personnel and in our experience works very well.
?. M-mode and two-dimensional measurements are used to assess systolic function.
?. An average of six selected cardiac cycles from at least two separate scans performed in randomblindfashion with papillary muscles used as a point of reference.
?. End diastole was defined as the maximal left ventricular (LV) diastolic dimension and end systole was defined as the peak of posterior wall motion.
?. Fractional shortening (FS), a surrogate of systolic function, was calculated from LV dimensions as follows: FS = ((EDD − ESD) / EDD) × 100%, where EDD and ESD are LV end diastolic and end systolic dimension, respectively.
TITLE: Bovine satellite cell Pax7 ICC
AUTHORS: Andrew Stout, Natalie Rubio

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Staining primary bovine satellite cells for Pax7, a common marker of satellite cells and myogenic potential. Protocol developed for </span><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.thermofisher.com/antibody/product/PAX7-Antibody-Polyclonal/PA5-68506</span></a><span> (Thermo Fisher Pax7 antibody PA5-68506; rabbit IgG anti-Pax7)</span></div></div>

[STEPS]
?. [Fixation and Permeabilization (1 hour)]
Aspirate media from cells
?. [Fixation and Permeabilization (1 hour)]
add cold 4% PFA to cells (enough to cover cells or scaffolds)
?. [Fixation and Permeabilization (1 hour)]
Incubate at room temperature for 30 minutes
?. [Fixation and Permeabilization (1 hour)]
Wash 3x with room temperature PBS
?. [Fixation and Permeabilization (1 hour)]
Aspirate PBS and add cold Permeabilization solution for 15 minutes
?. [Fixation and Permeabilization (1 hour)]
Wash 3x with cold PBST
?. [Primary Stain (1 hour, overnight incubation)]
Aspirate PBST and add cold Wash buffer for 45 minutes
During soak, can move to step 8
?. [Primary Stain (1 hour, overnight incubation)]
Dilute primary antibodies in wash buffer and keep on ice (protected from light). For given antibody, use the following dilutions:anti-Pax7 (1:500)Phalloidin-594 (1:100)note* prepare enough antibody solution for all conditions (a little extra is usually good to make sure there is enough)
?. [Primary Stain (1 hour, overnight incubation)]
After step 7 incubation, wash 3x with cold PBST
?. [Primary Stain (1 hour, overnight incubation)]
Add primary antibody solutions and incubate overnight at 4C (parafilm to avoid evaporation)
?. [Secondary Stain (1.5 hours)]
Wash 3x with cold PBST
?. [Secondary Stain (1.5 hours)]
Aspirate PBST and add cold Wash buffer for 15 minutes
during soak, can move to step 13
?. [Secondary Stain (1.5 hours)]
Dilute secondary antibodies in wash buffer and keep on ice (protected from light). For given antibody, use the following dilutions:note* prepare enough antibody solution for all conditions (a little extra is usually good to make sure there is enough)
?. [Secondary Stain (1.5 hours)]
After step 12 incubation, aspirate Wash buffer from cells, and add secondary antibody solutions. Incubate in the dark at room temperate for 60 minutes
?. [Secondary Stain (1.5 hours)]
Wash cells 3X with cold PBST, leaving the last wash to soak for 5 minutes
?. [Secondary Stain (1.5 hours)]
Aspirate PBST, and add DAPI mounting media. Cover with cover-slip, and image after 10 minutes Pax7 = greenActin cytoskeleton = rednuclei = blue
TITLE: Use of cholera toxin subunit B to label neural projections to major pelvic ganglia
AUTHORS: Janet R Keast, Peregrine B Osborne, Nicole Wiedmann, John-Paul Fuller-Jackson

[DESCRIPTION]
This protocol is used to visualise preganglionic neurons in the spinal cord innervating pelvic visceral organs (e.g., the lower urinary tract) in an experimental adult male or female rat. The protocol is performed under anesthesia and should incorporate all local requirements for standards of animal experimentation, including methods of anesthesia, surgical environment, and post-operative monitoring and care.

[STEPS]
SECTION: Preparation for surgery
1.  Prepare cholera toxin subunit B solutions: low salt formulation with 0.05% Evans Blue.
SECTION: Preparation for surgery
3. Anesthetise animal (2.5% isoflurane in oxygen, or as required for maintenance)
SECTION: Preparation for surgery
4.  Apply eye lubricant and place animal on heated pad.
SECTION: Preparation for surgery
5.  Shave and clean the ventral abdomen.
SECTION: Surgery
6. Perform a midline incision in the skin and then the muscle, then gently move organs to visualise the required injection site.

The major pelvic ganglia is located on the dorsal lateral lobe of prostate (male) or cervix (female)
SECTION: Surgery
8. Microinject sterile tracer solution at the selected injection site using a glass pipette attached to a picospritzer. At each injection site, hold the glass pipette in place for ~5 seconds after ejection of the dye, to enable the dye to spread to the underlying tissue. This also minimises leakage. Continue injections until the desired volume is reached.


SECTION: Surgery
9. Wash all injection sites with sterile saline.
SECTION: Surgery
11. Close the muscle and skin using approved procedures. Administer analgesics and monitor animal during postoperative period as per local approved procedure. 
SECTION: Tissue harvesting
12. To analyse tracer distribution in spinal cord or the injection site, 3 days after surgery, anesthetise animals as per local ethical requirements, and perform intra-cardiac perfusion with fixative, then dissect tissues of interest for further study.


SECTION: Surgery
7. Using fine angled forceps, gently blunt dissect underneath the pelvic ganglia. Once the ganglia is separated from the underlying tissue, slide a sterile 2mm x 2mm piece of parafilm between it and the tissue.
SECTION: Surgery
10. For a bilateral injection, perform steps 6-8 on the alternate side.
SECTION: Preparation for surgery
2. Prepare glass pipettes for surgery by prefilling each pulled glass pipette with cholera toxin subunit B
TITLE: Ethanol precipitation of small DNA fragments
AUTHORS: Eva Petrova, Roey Angel

[DESCRIPTION]
This protocol is for a simple ethanol precipitation of small fragments and/or small amounts of DNA. Note that this protocol simply concentrates your sample and removes enough salts/enzymes for ligation to be successful. All DNA fragments from your digest will still be present in your pellet.
 See here for a discussion of theory behind ethanol precipitation of DNA fragments.
 

[BEFORE_START]
Absolute Ethanol (100% = 200 proof) at -80o C
70% ethanol at -80o C (don’t cool ethanol for too long or water will freeze)
Tabletop centrifuge
-80°C freezer

[GUIDELINES]
3M Sodium Acetate, 0.11M MgCl2 pH 5.5
24.6 g sodium acetate
0.952g MgCl2 anhydrous
Dissolve in about 60 ml DDW Adjust pH with glacial acetic acid (>8 ml), Dilute to 100 ml with DDW and filter sterilize.
 

[STEPS]
1. Transfer the sample to 1.5 mL eppendorf tube.
2. Adjust the salt conc. of the sample to 0.3M sodium acetate and 0.01M MgCl2.
0.3 21
0.01 21
3. Add 1 µl of Pellet Paint NF.
1 1
 
4. Add 1 µl of non labeled genomic DNA e.g. from E.coli (this acts as a carrier and increases precipitation yield).
1 1
5. Add 2 volumes cold absolute ethanol to sample.
6. Incubate 2-3 hr at -80°C.  The long incubation time is critical for small fragments.
-80 10
120 min
7. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
 
0 10
30 min
8. Remove supernatant.
9. Wash with 750-1000 μL cold 70% ethanol.  Another critical step for small fragments under 200 base pairs. Washing involves adding the ethanol and inverting several times.
750 1
10. Centrifuge for 30 minutes at 0°C at maximum speed (generally >10000 g at least).
 
0 10
30 min
11. Repeat washing procedure and centrifuge again.
   
12. Let air dry on benchtop.
13. Resuspend in an appropriate volume of H2O.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
PCPipe is a protein-clustering tool.  The input is a set of ORFs and a FASTA file with already clustered ORFs. <br /><br />The process entails:<br />
<ul>
<li>Use <a href="http://weizhongli-lab.org/cd-hit/" target="_blank">cd-hit-2d</a> to compare the input peptides to previously clustered proteins</li>
<li>The result is a file with input proteins that clustered to existing clusters and those that did not</li>
<li>Use the unclustered peptides and self-cluster them via <a href="http://weizhongli-lab.org/cd-hit/" target="_blank">cd-hit</a></li>
<li>Take a representative sequence from each novel cluster, and use "<a href="https://www.ncbi.nlm.nih.gov/books/NBK279690/" target="_blank">blastp</a>" to compare to <a href="http://cube.univie.ac.at/resources/simap" target="_blank">SIMAP</a>.</li>
<li>Use the resulting SIMAP "feature_id" to look up the <a href="http://fileshare.csb.univie.ac.at/simap/" target="_blank">SIMAP features</a>, merging the query results with the protein ID into a tab-delimited annotations file</li>
<li>Provide the user with two cluster files and the annotations for the new clusters based on the representative sequence</li>
</ul>
<br />Code is freely available at <a href="https://github.com/hurwitzlab/pcpipe" target="_blank">Github</a>.

[STEPS]
?. 
?. 
?. 
?.
TITLE: Immunocytochemistry
AUTHORS: Oriol Busquets, Hanqin Li, Khaja Mohieddin Syed, Riana Lo Bu, Frank Soldner, Dirk Hockemeyer

[DESCRIPTION]
This protocol summarizes the steps to immunostain hPSCs or differentiated cultures.

Protocol Overview
A. Cell fixation
B. Immunocytochemistry

[STEPS]
SECTION: Cell fixation
1. 
 

 

Remove the media and gently rinse the cells with 1X PBS. Remove PBS and add 4% PFA in PBS. Incubate the cells at Room temperature (RT) for 15 min to 20 minutes.
SECTION: Cell fixation
2. After fixation, discard the PFA and wash cells with 1xPBS at Room temperature for 3 times 10 min each. 
SECTION: Cell fixation
3. At this stage, samples may be stored at 4 °C for up to a week before continuing with the staining process. It is recommended to seal with parafilm to prevent evaporation and store the plate in the dark.
SECTION: Immunocytochemistry
4. 
 

 

Permeabilization (optional for surface proteins): add 0.3% Triton X-100 in PBS  and incubate the cells for 15 min to 20 minutes at Room temperature.
SECTION: Immunocytochemistry
5. Wash the cells three times with 1xPBS at room temperature for 10 min each.
SECTION: Immunocytochemistry
6. Blocking: Add blocking solution (10% serum or 3% BSA in PBS) and incubate for at least 60 min at Room temperature. Note: source/species of serum used in blocking solution must be compatible with host species of the primary antibody.
SECTION: Immunocytochemistry
7. Remove the blocking solution and add the primary antibody solution. Primary antibody/ies are diluted in blocking solution to the appropriate concentration.
SECTION: Immunocytochemistry
8. Incubate the plate at 4 °C 60 min.
SECTION: Immunocytochemistry
9. Remove the primary antibody solution and wash the cells with 1xPBS for three times 10 min each at Room temperature.
SECTION: Immunocytochemistry
10. Add the secondary antibody/ies diluted in PBS to the appropriate concentration. Incubate for 60 min at Room temperature in the dark. Note: all subsequent steps after the addition of the secondary antibody should be performed under limited light exposure of samples to light to prevent fluorescence bleaching.
SECTION: Immunocytochemistry
11. Remove the secondary antibody solution and wash the cells with 1xPBS for three times 10 min each at Room temperature.
SECTION: Immunocytochemistry
12. Add DAPI diluted in 1xPBS and incubate the cells at RT for 5 min to 10 minutes.
SECTION: Immunocytochemistry
13. Rinse the cells three times with 1xPBS. Samples can be stored in the fridge until they are imaged directly from the plate or mounted onto microscope slides for imaging.
SECTION: Immunocytochemistry
14. Mounting: add a drop of ProLong‱ Glass Antifade Mountant on a microscope slide and place the coverslip on top avoiding bubble formation. Keep in the dark until mountant has dried and image directly or store in the fridge until use.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Kit Components</strong></p>
<p><br />• Capture Antibody Coated Plate* - 1 plate<br />• a-Synuclein Standard - (1) 25μg vial<br />• 5X Wash Buffer - 250mL<br />• 2X Reagent Diluent - 32mL<br />• Biotinylated Primary Antibody - 25μL<br />• Streptavidin-HRP - 25μL<br />• Chemiluminescent Substrate A - 6mL<br />• Chemiluminescent Substrate B - 6mL<br />• Plate Sealer - 3</p>
<p> </p>
<p><em>* The strips in this plate are not readily removable; it is intended to be used as a whole plate. Attempting to remove </em><em>the strips will result in damage for which Biolegend is not responsible.</em></p>
<p><strong> </strong></p>
<p><strong>Storage:</strong> Store kit at 2-8oC. We suggest storing the a-Synuclein Standard component only at ≤-70oC if you do not intend to use the kit within 2 weeks of receipt.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dt9ecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dt8ecje.jpg" data-ofn="alpha1.JPG" /></p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dubecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/duaecje.jpg" data-ofn="alpha2.JPG" /></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dudecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/ducecje.jpg" data-ofn="alpha3.JPG" /></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dufecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dueecje.jpg" data-ofn="alpha4.JPG" /></p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dujecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/duhecje.jpg" data-ofn="alpha5.JPG" /></p>

[STEPS]
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TITLE: U Mass - Creatinine
AUTHORS: Jason Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">
This experiment involves a spectrophotometric measurement using Roche Cobas Clinical Chemistry Analyzer. Serum levels of creatinine reflect systemic protein metabolism.</div></div>

[STEPS]
?. Perform daily quality control assessment of instrumentation before analysis.
?. Load each sample into a specialized micro-sample cup for the clinical chemistry analyzer.
?. Select Creatinine test on display and run the analysis.
?. Collect and analyze the data.
TITLE: LC3 Lipidation assay for ATG3 Mutants
AUTHORS: lmstrong

[DESCRIPTION]
LC3 lipidation assay on liposomes 

[STEPS]
1. Mix 1 μM ATG3 WT or mutant, 1 μM ATG7, 500 nM WIPI2d, 1 μM ATG12–ATG5-ATG16L1, 5μM LC3B 
and  0.5 mg/mL extruded liposomes in reaction buffer (25mM HEPES pH 7.4, 150 mM NaCl, 1 mM TCEP).
2. Incubate at room temperature for 20 minutes. 
3. Quench with 4X LDS loading buffer. Boil samples for 10 minutes at 60ºC. Load and run SDS PAGE gel
4. Visualize protein bands with coomassie blue
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Isolation of plasmid DNA from E. coli using the alkaline lysis method modified from Birnboim <em>et al.</em>, 1979. </p>
<p>This protocol is suitable for fast, cheap recovery of large amounts of plasmid, e.g. for cloning purposes or restriction analysis. However, the purity of plasmid is insufficient for sequencing.</p>

[STEPS]
?. 
?. 
?. 
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TITLE: Protocol 1*
AUTHORS: Mitt Coats

[DESCRIPTION]
A short abstract

[STEPS]
3. 
 

SECTION: S2
4. 
 

SECTION: S2
5. 
 

SECTION: S2
6.   

SECTION: S0
1. 
SECTION: S1
2. 
 

SECTION: S2
7. Done  

SECTION: S2
8. Yolo
TITLE: Protocol for the production and storage of Diplodia sapinea pycnidiospores
AUTHORS: Anne Geertje Oostlander, Laura Brodde, Miriam von Bargen, Rasmus Enderle, Marco Leiterholt, Dagmar Trautmann, Malin Elfstrand, Jan Stenlid, André Fleißner

[DESCRIPTION]
An efficient, standardized protocol for the production and storage of highly viable pycnidiospores of Diplodia sapinea. 

[STEPS]
1. Inoculate a 5.5 cm Petri dish containing 10 to 15 ml of solid VMM with D. sapinea with an agar plug with mycelia from a preculture on VMM.
2. Incubate the plate for 21 d at 26 to 29°C with constant light (cold daylight at an intensity of 5000 to 650 lux). Pycnidia will form on the surface of the culture and open to release a droplet of liquid containing spores.
3. Harvest the spores from the plate by adding approximately 2 ml of 0.01% (v/v) Tween to the surface and rinse the surface of the colony several times by pipetting. Repeat the procedure for a higher yield. Typically, between 1x106 and 2x106 spores can be harvested per plate. Spores can be separated from the liquid by centrifugation for 10 s at 5000 rpm and then resuspended in your desired volume.
4. For storage, mix 500 µl of the spore suspension and 500 µl 40% glycerol in a 2 ml screw top tube or cryovial. Freeze the glycerol stock tube at -80°C.
TITLE: Constant Potential Amperometry in vitro
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol for Constant Potential Amperometry recordings in vitro of evoked dopamine release in the rat striatum

[STEPS]
SECTION: Slice preparation
1. Anesthetize the animal by isofluorane inhalation and decapitate the animal. 
SECTION: Slice preparation
2. Revome quickly and carefully the brain from the skull, and slice the brain in 250–300 µm thick coronal slices containing the striatum by a vibratome immersed in cooled bubbled (95% O2, 5% CO2) ‘sucrose-based’ artificial CSF (aCSF) solution containing (in mM): KCl3, NaH2PO4 (1.25), NaHCO3 (26), MgSO4 (10), CaCl2 (0.5), glucose (25), sucrose (185); ~300 mOsm, pH 7.4 
SECTION: Slice preparation
3. Transfer slices in standard aCSF solution containing (in mM): NaCl (126), KCl (2.5), NaH2PO4 (1.2), NaHCO3 (24), MgCl2 (1.3), CaCl2 (2.4), glucose 10; ~290 mOsm, pH 7.4, bubbling (95% O2, 5% CO2); at 32 °C, and let them recovery for at least 40 min
SECTION: Amperometric recordings
5. Place a bipolar Ni/Cr insulated stimulating electrode into the striatal slice. To monitor the
electrically evoked dopamine overflow, gently place a carbon fiber electrode (active surface 30 μm in diameter and 100 μm long; World Precision Instruments, CF10) into the striatal slice to a depth of 100–150 μm near the stimulating electrode, connected to a potentiostat (MicroC, World Precision Instruments) to apply voltage and measure current.
SECTION: Amperometric recordings
6. Apply every 5 min a single rectangular electrical pulse through a bipolar stimulating electrode placed on the striatal slice by using a Digitimer DS3 stimulator (80–500 μA, 20–40 μs duration)
SECTION: Slice preparation
4. Transfer one slice for amperometric recordings into the recording chamber under an optical microscope, and superfuse (2-3 ml/min) the slice with standard aCSF solution kept at 32°C
SECTION: Amperometric recordings
5.1.  Impose 0.55 V between the carbon fiber electrode and the Ag/AgCl pellet 
SECTION: Amperometric recordings
6.1. Apply increasing electrical pulse step until maximum evoked dopamine outflow 
SECTION: Drug application
8. Monitor the extracellular dopamine response to electrical stimulation is stable for four or five successive stimulations
SECTION: Amperometric recordings
7. At the end of each experiment, perform electrode calibration in aCSF containing known dopamine concentration (300 nmol/L–3 μmol/L)
SECTION: Drug application
9. Superfuse the striatal slice with drugs (0.3 µM cocaine, 1 µM cocaine, or 0.03 µM quinpirole, dissolved in standard aCSF) for 10 min (cocaine) and 5 min (quinpirole) 
SECTION: Drug application
9.1. Wash out with standard  aCSF for 1h (cocaine) or 30 min (quinpirole) 
SECTION: Drug application
10. At the end of each experiment, perform electrode calibration in aCSF containing known dopamine concentration (300 nmol/L–3 μmol/L)