eamag/protocols-with-steps huggingface dataset:
TITLE: Levodopa kinetic-dynamic test
AUTHORS: Manuela Contin 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;"> Levodopa oral test </span></div><div class = "text-block">Administration of a subacutest dose of levodopa (100 mg) plus benserazide (25 mg) or carbidopa (25 mg) under standardized conditions.</div><div class = "text-block"><span style = "font-weight:bold;">Levodopa kinetic–dynamic monitoring</span><span></span></div><div class = "text-block">Measurements of plasma levodopa concentrations and clinical effects by objective motor tests (alternate finger tapping test). </div></div>

[STEPS]
?. Blood samplings are drawn by an indwelling catheter before levodopa dosing, at 15-min intervals for the first 90 min, then half-hourly, up to 3 hours after dosing.
?. Blood specimens are transferred into heparinized tubes and immediately centrifuged at 1500g for 10 minutes at 4°C. Plasma samples are stored at −80°C until analysis (usually within 6 months).
?. Levodopa plasma concentration measurements are performed by high-performance liquid chromatography (HPLC) with coulometric detection (Baruzzi, A., Contin, M., Albani, F., and Riva, R. (1986) Simple and rapid micromethod for the determination of levodopa and 3-O-methyldopa in human plasma by high-performance liquid chromatography with coulometric detection. J Chromatogr, 375:165-169).
?. Motor effects are tested by the alternate index finger tapping test (the number of times the patient could alternately tap two buttons 20 cm apart in 60 seconds with the most affected hand) up to 4 hours post-dosing, using a computerized touch screen system (mTapping, mHealth Technologies srl, Bologna, Italy).
?. Possible levodopa induced dyskinesias are rated at the same times as motor responses by the Clinical Dyskinesia Rating Scale (CDRS) (Hagell P., Widner H. (1999) Clinical rating of dyskinesias in Parkinson’s disease: use and reliability of a new rating scale. Mov Disord, 14: 448-455.).
?. Administration of a subacute test dose of levodopa (100 mg) plus benserazide (25 mg) or carbidopa (25 mg) under standardized conditions:- Fasting dosing- At least 12 hours of levodopa and possibly concomitant antiparkinsonian therapy wash-out
TITLE: Deepwell Reusual Protocol 
AUTHORS: Morris Jack

[DESCRIPTION]
This is a protocol to reuse deepwells for sustainability practices

[STEPS]
SECTION: Growing cultures in a deep well
1. Cover a deep well with two layers of aluminium foil and tape the front and back to hold it securely. The tape also turns the aluminium foil into a lid for the deep well by temporarily peeling off the front tape.
SECTION: Growing cultures in a deep well
2. Autoclave at 120C for 20min. After autoclaving, keep in a heated oven (> 50C) overnight for drying and desiccation.
SECTION: Growing cultures in a deep well
3. Do not fill the deep wells more than 60% of their max volume if they are to be placed in a shaking incubator to avoid spillage.
SECTION: Deep well Decontamination
4. Tape around the deep well over the aluminium foil to prevent spillage.
SECTION: Deep well Decontamination
5. While carrying the deep well outside the lab space, enclose it in a waterproof autoclave bag. Keep sufficient absorbent paper on hand in case of accidental spillage.
SECTION: Deep well Decontamination
6. In the autoclave facility, autoclave the deep wells at 120C for >30min to decontaminate.
SECTION: Deep well Decontamination
7. Once autoclaved, the deep wells will be brought back to the lab
SECTION: Deep well Decontamination
8. Carefully peel the aluminium foil and dispose the cultures and aluminium foil into a disposal bag containing paper roll to absorb the liquid.
SECTION: Deep well Decontamination
9. Send decontaminated culture for incineration (yellow bin route).
SECTION: Deep well Decontamination
10. Rinse the deep well plate with water and a brush to clean before future use.
SECTION: Deep well Decontamination
11. Keep in a heated oven (> 50C) overnight for drying and desiccation
SECTION: Major Risks
12. Using deep wells presents similar risk as using a microtiter plate and similar precautions need to be taken.
SECTION: Major Risks
13. Spilling of culture during shaking - At speeds of 180rpm, the spillage is be negligible
SECTION: Major Risks
14. Spillage while handling - If handled with reasonable care and awareness, the risk of spillage is negligible. Presence of the aluminium foil and tape reduces this risk. Even if inverted, outside spillage would be minimal, however care would have to be taken while removing the aluminium foil lid post autoclaving.
TITLE: DoiClassesOfErrors
AUTHORS: Ricarda Boente, Deniz Tural, Cristian Santini, Arcangelo Massari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to describe an automated process to repair invalid DOIs. In particular, four classes of errors are addressed: previously invalid DOIs become valid, prefix errors, suffix errors, and other type errors.</div></div>

[STEPS]
?. [Checking  the DOI names' invalidity]
First, we check for each cited DOI if it is factually invalid. For this purpose, the DOI Proxy is used (https://www.doi.org/factsheets/DOIProxy.html): if the status code corresponding to that specific DOI is different from 1, it means that it is not valid; otherwise, that DOI has become valid in the meanwhile and the algorithm returns the same DOI as a correct one.
?. [Error analysis]
Taking as a reference the DOI error's taxonomy by Buchanan (Buchanan, 2006), there are two main classes of errors: author errors, made by authors when creating the list of cited articles for their publication, and database mapping errors, related to a data-entry error. This protocol deals only with the second kind of error, which can be further divided between prefix errors, suffix errors, and other-type errors (Xu et al., 2019). In order to solve our problem, we isolated recurrent strings at the beginning and at the end with corrupted DOI prefix and suffix respectively. In addition, we found other types of errors, like wrongly encoded characters and unwanted characters, that could be removed at the end of the data cleaning process.
?. [Data cleaning process]
Here we describe the steps through which we carried out the cleaning process of factually invalid DOIs
?. [Data cleaning process]
We define two regular expressions: one for identifying corruptions at the beginning of the DOI and one for cleaning corruptions at the end.
?. [Data cleaning process]
For each factually invalid DOI, we apply the two regular expressions for cleaning prefix-type and suffix-type errors
?. [Data cleaning process]
Once we have cleaned the DOI from the aforementioned error types we remove unwanted characters, that is double underscores, double periods, XML tags, spaces, and forward slashes.
?. [Data cleaning process]
After this procedure, we store the modified DOIs in an output CSV file, where for each cited DOI we also store a value corresponding to the error class to which it belongs.
?. [Checking the modified DOI names]
Finally, each modified DOI name is checked through the DOI Proxy, to verify if the aforementioned procedure was able to fix it or not. In the end, we provide the number of DOI names that we were able to fix and the number of DOI names for each class of errors.
?. [Checking  the DOI names' invalidity]
We import a CSV containing invalid DOI-to-DOI citations organized as follow: the first column contains valid citing DOIs and the second contains invalid cited DOIs
TITLE: ChAT and human alpha-synuclein immunofluorescence staining
AUTHORS: Pietro La Vitola

[DESCRIPTION]
This protocol is designed for detecting choline acetyltransferase (ChAT) using the Millipore AB144P (RRID:AB_2079751) and human alpha-synuclein (Abcam, RRID:AB_2537217, MJFR1). Tissues stained with this protocol include 35 μm free-floating mouse brain sections. All tissues were from mice perfused with 4% PFA. 


[STEPS]
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
1. Wash tissue slices in tris-buffer saline (TBS: 0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat x3.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
4. Incubate in blocking buffer (5% donkey serum, 2% BSA, 0.5% triton X-100 in TBS) for 1 hour at room temperature.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
6. Wash tissue slices in TBS-T (TBS+ 0.25% Triton x-100) for 10 minutes. Repeat x3.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
7. Incubate in fluorescent secondary antibody at 1:200 dilution in 50% blocking buffer for 1 hour at room temperature.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
8. Wash tissue slices in TBS-T(TBS+ 0.25% Triton X-100) for 10 minutes. Repeat x 2.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
10. Mount free-floating sections on SuperFrost+ slides (if staining free-floating tissue) and let dry at room temperature for 15 minutes.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
11. Coverslip with fluorescent mounting medium and #1.5 coverslips. Outline coverslip with clear nail polish and store at 4oC .
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
5. Incubate with  anti-ChAT primary antibody  (RRID:AB_2079751) at 1:500 dilution and anti-hASYN (1:10000; RRID:AB_2537217, MJFR1 in 50% blocking solution for 48 hours at 4°C. 
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
2. Incubate in in 10% Methanol + 3% H2O2 in TBS for 20 min at room temperature
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
9. Wash tissue slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes.
SECTION: ChAT and human alpha-synuclein immunofluorescence staining
3. Wash tissue slices in tris-buffer saline (TBS: 0.05M Trizma base and 0.15M NaCl; pH: 7.6) for 10 minutes. Repeat x3.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Soil- and sensor specific calibration of Sentek EnviroSCAN and ML3 sensors for the Lonzée experimental site.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>Note: If the percentage of CD45+ cells in your sample is less than 50%, please follow Protocol A. If it is higher </strong></p>
<p><strong>than 50% then please follow protocol B.</strong></p>
<p> </p>
<p>The cells targeted by the Nanobeads are either selected or depleted by incubating your sample with the directly conjugated magnetic particles. The magnetically labeled fraction is retained by the use of a magnetic separator. After collection of the targeted cells, downstream applications include functional assays, gene expression, phenotypic characterization, etc.</p>

[GUIDELINES]
<p><strong>Protocol:</strong> This procedure is optimized for the isolation of 10<sup>7</sup> to 2 x 10<sup>8</sup> cells per tube. If working with fewer than 10<sup>7</sup> cells, keep volumes as indicated for 10<sup>7</sup> cells. For best results, optimize the conditions to your specific cell number and tissue. Prepare fresh MojoSort™ Buffer solution by diluting the 5X concentrate with sterile distilled water.</p>
<p> </p>
<p><strong>Reagents and instruments required:</strong></p>
<p> </p>
<p>-MojoSort™ Buffer (5X) (Cat. No. 480017)<br />-MojoSort™ Magnet (Cat. No. 480019) or compatible magnetic separation system<br />-Adjustable pipettes<br />-70 μm filters (one per sample)<br />-5 mL (12 x 75 mm) polystyrene tubes<br />-Reagents for sample preparation<br />-Reagents and instruments (Flow cytometer) to determine yield and purity</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/dpiecje.jpg" data-src="https://s3.amazonaws.com/pr-journal/dphecje.jpg" data-ofn="MojoSort_humanCD45protocolA.JPG" /></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
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?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The Internal Transcribed Spacer 2 (ITS2) rRNA gene is a commonly targeted genetic marker to assess diversity of <em>Symbiodinium</em>, a dinoflagellate genus of algal endosymbionts that is pervasively associated with marine invertebrates, and notably reef-building corals. Here we tested three commonly used ITS2 primer pairs (SYM_VAR_5.8S2/SYM_VAR_REV, ITSintfor2/ITSReverse, and ITS-DINO/ITS2Rev2) with regard to amplification specificity and sensitivity towards <em>Symbiodinium</em>, as well as sub-genera taxonomic bias<em>.</em> We tested these primers over a range of sample types including three coral species, coral surrounding water, reef surface water, and open ocean water to assess their suitability for use in large-scale next generation sequencing projects and to develop a standardized PCR protocol. We found the SYM_VAR_5.8S2/SYM_VAR_REV primers to perform superior to the other tested ITS2 primers. We therefore used this primer pair to develop a standardised PCR protocol. To do this, we tested the effect of PCR-to-PCR variation, annealing temperature, cycle number, and different polymerase systems on the PCR efficacy. The <em>Symbiodinium</em> ITS2 PCR protocol developed here delivers improved specificity and sensitivity towards <em>Symbiodinium</em> with apparent minimal sub-genera taxonomic bias across all sample types. In particular, the protocol’s ability to amplify <em>Symbiodinium</em> from a range of environmental sources will facilitate the study of <em>Symbiodinium</em> populations across biomes.</p>

[STEPS]
?.
TITLE: Abeoforma whisleri transient transfection protocol
AUTHORS: Elena Casacuberta, Aleksandra Kozyczkowska, Sebastián Najle

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for transient transfection of the Icthyosporean Abeoforma whisleri. This protocol has an efficiency of 1-2% calculated from fluorescent positive cell from Flow Cytometry data, using the amount of cells and DNA described in the protocol. Increasing DNA from above 1 microgram/microliter does not increase transfection.</div></div>

[STEPS]
?. [Pre-Transfection]
Count A.whisleri cells from a culture 1 to 2 weeks old grown in Marine Broth (MB) medium at 13 degrees.
?. [Pre-Transfection]
Take 2 x 105 cells/per transfection condition, and spin them down at 2000g for 5 min.
?. [Pre-Transfection]
Discard medium and gently resuspend cells in sterile 1xPBS and spin them down again (same conditions as above)
?. [Transfection]
Discard PBS and resuspend cells with 20-25 microliters of mixture Buffer P3 Lonza+1 to 5 micrograms of reporter plasmid+ 40 micrograms of carrier DNA (empty vector such as pUC19).
?. [Transfection]
Transfer de 20-25 microlters mixture to a 16 well strips from Lonza Nucleofector (X Unit) and apply CODE EN-138.
?. [Post-Transfection]
Inmediately after transfection add 80 microliters of MB directly to the well. You can transfer directly to a 6 well plate with 1ml of media, or wait for 30 minutes.
?. [Post-Transfection]
Screen for transformants after 24 hours of transfection, the number of positive cells will increase untill 48 hours.
?. [IMPORTANT]
Include always a well of cells with no DNA. A.whisleri shows autofluorescence, so this control is key to compare and decide what is real transfection and what is autofluorescence.Use high quality DNA and as concentrated as possible. We liofilize DNA prep and resuspend them to have a concentration of at least 1.5 micrograms/microl.Use ultrapure and concentrated carrier DNA. We use pUC19 at 20micrograms/microliter.
TITLE: Colorimetric determination of urea
AUTHORS: noah.langenfeld , Laurenpayne , Bruce Bugbee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol measures the absorbance of urea in solution in complexation with diacetyl monoxomie at 520 nm and is linearly proportional to concentration up to 5 mM urea.</div></div>

[STEPS]
?. [Mixed Acid Reagent Preparation]
Dissolve   ferric chloride in   deionized water in a 250 mL volumetric flask.
2.5 mg
45 mL
?. [Mixed Acid Reagent Preparation]
Add  phosphoric acid.
80 µl
?. [Mixed Acid Reagent Preparation]
Prepare  sulfuric acid by diluting  concentrated sulfuric acid up to  with deionized water.
Caution: Preparation of    sulfuric acid is highly exothermic. Slowly add acid to   water and stir frequently to avoid spattering.
65.25 mL
250 mL
?. [Mixed Acid Reagent Preparation]
Dilute ferric chloride, deionized water, and phosphoric acid mixture to  with  sulfuric acid.
250 mL
?. [Mixed Acid Reagent Preparation]
Mix until dissolved.
?. [Mixed Color Reagent Preparation]
Add   diacetyl monoxime and  thiosemicarbazide to a 250 mL volumetric flask.
20.9 mg
52.4 mg
?. [Mixed Color Reagent Preparation]
Dilute to volume () with deionized water.
250 mL
?. [Mixed Color Reagent Preparation]
Mix until dissolved.
?. [Urea Assay]
Fill a 600 mL beaker with  deionized water.
200 mL
?. [Urea Assay]
Place the 600 mL beaker on hot plate and bring to a boil.
?. [Urea Assay]
To test a sample, aliquot  into a 20 mL glass test tube.
1 mL
?. [Urea Assay]
Add Mixed Acid Reagent.
2 mL
?. [Urea Assay]
Add  Mixed Color Reagent.
2 mL
?. [Urea Assay]
Ensure contents of test tube are vigorously mixed by vortexing for at least  .
?. [Urea Assay]
Repeat steps 12-15 for each sample and standard to be analyzed.
?. [Urea Assay]
Ensure test tubes are labeled and place in boiling water bath for  .
?. [Urea Assay]
Remove tubes from water bath and let cool for  .
?. [Urea Assay]
Fill plastic cuvette to line with cooled solution.
?. [Urea Assay]
Place sample cuvette in spectrophotometer and record absorbance at 520 nm. Blank against the standard solution with both reagents, but no urea.
?. [Urea Assay]
Prepare known concentrations of urea at , , ,, , and  for a calibration curve.
?. [Urea Assay]
Use the calibration curve to determine urea concentrations of unknown solutions.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Este protocolo describe los pasos para propagar <em>in vitro</em> hongos tipo <strong>sombrero</strong> (Ver <a href=" https://www.protocols.io/private/be76339cd7204260002c2492d08d30cf" target="_blank">este</a> protocolo para hongos tipo <strong>terraza</strong>). Este protocolo ha sido desarrollado bajo la guia de Phil Ross (Mycoworks; https://www.mycoworks.com).</p>
<p> </p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/tyfeme6.jpg" data-src="https://s3.amazonaws.com/pr-journal/tyeeme6.jpg" data-ofn="20170402_125510_hdr.jpg" /></p>
<p> </p>

[BEFORE_START]
<p>Al muestrear se debe recolectar todo el cuerpo fructífero que sea posible, cuidando estípite y volva del hongo.  </p>
<p>Las muestras deben ser cultivadas lo antes posible luego de su recolección para aumentar las probabilidades de éxito. Si bien la duración del hongo antes de su descomposición dependerá de las condiciones de almacenaje y el hongo en sí, se recomienda realizar los cultivos dentro de las 24 horas siguientes a la recolección. </p>
<p> </p>
<p>El cultivo de las muestras debe hacerse en condiciones de esterilidad.</p>
<p>Finalmente, cabe recordar que si bien, muchos de los hongos que se puedan recolectar son cultivables por este método, existen varios que serán difícilmente cultivable o no podrán ser cultivados. Dado lo anterior, es importante sacar siempre varias muestras y seguir los pasos con la mayor precisión posible para aumentar las probabilidades de éxito.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: SPARC Cat - Sham Control Chronic Cat 1, Day 14
AUTHORS: Brett   Hanzlicek, Anna Rietsch, Margot Damaser

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a procedure for a sham control chronic cat experiment (Day 14) for cystotomy (bladder surgery).  The cystotomy is performed without UroMOCA implantation.  The cat is observed daily and imaged on day 0, 14 and 30 to track changes to the bladder and overall cat health in response to the cystotomy.  This protocol includes urodynamics and imaging for Day 14 in the chronic experiments.</div></div>

[STEPS]
?. [Transport Cat]
Transport cat from housing site to surgery site.
?. [Animal Prep and catheter placement]
Animal is anesthetized and abdomen is shaved by the vet team.  The cat is then moved into the surgery room and attached to monitors by the vet team.
?. [Animal Prep and catheter placement]
Drape animal and perform betadine scrub on abdomen and genitals.
?. [Animal Prep and catheter placement]
Put surgilube on 3.5Fr catheter and insert into bladder through the urethra.  Advance the catheter until resistance is met, then pull back 2-3cm.
?. [Animal Prep and catheter placement]
Use gentle suction with syringe to withdraw urine from bladder through the catheter.  Measure the volume and save the urine for urinalysis.  urine removed
16 mL
?. [Cystogram - DYNA CT]
Use 1:5 dilution of contrast to saline to visualize bladder.Fill the bladder with   contrast and take a single CT image.Add   more to bladder (total of 15ml) and take single CT imageTake a 3D CT image with 15ml contrast:saline in the bladder
10 mL
5 mL
?. [Cystogram - DYNA CT]
Empty Bladder
?. [Urodynamics/Cystometry]
Performed after cat is transferred from Isoflurane to propofol anesthetic.
Fill rate is 2ml saline/minute using syringe pump.
Data is recorded using Labview software.
Pressure is recorded using an external pressure transducer connected to syringe pump on one end and tubing that leads to the catheter on the other end.
?. [Urodynamics/Cystometry]
Fill bladder with saline and record pressure using pressure transducer connected to  LabViewFill to  ;End round 1 urodynamics
28.6 mL
?. [Urodynamics/Cystometry]
Empty Bladder
?. [Urodynamics/cystometry 2]
Fill bladder with saline at 2ml/min
?. [Urodynamics/cystometry 2]
Fill to  ; End round 2 urodynamics
19 mL
?. [Wake and return cat]
Wait for cat to wake up and return to housing site.
TITLE: Antidepressant for Symptoms Remission of Gastroesophageal Reflux Disease: A Network Meta-Analysis (protocol)
AUTHORS: Xiaobei Si, Linyu Huo, Shuai Wang, Yu Lan, Shuo Zhang, Xumin Zhang

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Objective</span><span> To evaluate and compare symptoms remission of antidepressants for gastroesophageal reflux disease (GERD). </span><span style = "font-weight:bold;">Methods</span><span> We plan to includ randomized controlled trials (RCTs) that evaluated efficacy of antidepressants for GERD by searching the MEDLINE, EMBASE, Web of Science, China National Knowledge Infrastructure Database (CNKI), Chinese VIP Information (VIP), Chinese Medical Databases (CMB) and Wan-Fang databases from the starting date of database establishment till January 1st, 2019. Database will be searched with the search terms of "gastroesophageal reflux disease", "antidepressant", etc. ADDIS 1.16.8 will be adopted for network meta-analysis. We will complement node split analysis to test inconsistency. Rank probability will also be complemented for comparison among antidepressants.</span><span style = "font-weight:bold;"> Conclusion</span><span> Our network analysis may further evaluate and .compare symptoms remission of antidepressants for gastroesophageal reflux disease (GERD), which is considered to be more conducive to clinical treatment of GERD.</span></div></div>

[STEPS]
TITLE: Emergency Contacts
AUTHORS: Steven Wilhelm, Gary LeCleir

[STEPS]
?. AB1Knoxville Fire Department9112Knoxville Police9113Ambulance Service91145UT Police974-3111 (call 911 first)67Environmental Health & Safety974-50848Radiation Safety Dept.974-55809BioSafety Dept.       974-19381011Steve Wilhelm (PRINCIPAL INVESTIGATOR)865-405-88171213Gary LeCleir865-368-1516
AB1Knoxville Fire Department9112Knoxville Police9113Ambulance Service91145UT Police974-3111 (call 911 first)67Environmental Health & Safety974-50848Radiation Safety Dept.974-55809BioSafety Dept.       974-19381011Steve Wilhelm (PRINCIPAL INVESTIGATOR)865-405-88171213Gary LeCleir865-368-1516