eamag/protocols-with-steps huggingface dataset:
TITLE: Prepare NGM plates with Amphotericin B
AUTHORS: Research Cancer UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare NGM plates with amphotericin B</div><div class = "text-block"><span>From "</span><a href="https://www.thermofisher.com/order/catalog/product/15290026?SID=srch-srp-15290026" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.thermofisher.com/order/catalog/product/15290026?SID=srch-srp-15290026</span></a><span>":</span></div><div class = "text-block">"Amphotericin B is the generic version of Fungizone. "Fungizone" is a  trademark of E.R. Squibb & Sons, LLC. Amphotericin B prevents the  growth of fungi by causing an increase in fungal plasma membrane  permeability. Gibco Amphotericin B is used to prevent the contamination  of cell cultures by yeast and multicellular fungi. Gibco Amphotericin B  contains 250 µg of amphotericin B and 205 µg of sodium deoxycholate per  mL of distilled water. The recommended working concentration ranges from  0.25 to 2.50 µg/mL." </div></div>

[STEPS]
?. 
?. ABCD1Ingredients  Quantity2NGM Media  1L3Cholesterol 5mg/ml1ml41M CaCl21ml51M MgSO41ml61M KH2PO425ml7Petri dish30mmas required8 50mmas required9 90mmas required10Fungizone400 microlitres 1 microtubbe
ABCD1Ingredients  Quantity2NGM Media  1L3Cholesterol 5mg/ml1ml41M CaCl21ml51M MgSO41ml61M KH2PO425ml7Petri dish30mmas required8 50mmas required9 90mmas required10Fungizone400 microlitres 1 microtubbe
?. A1First thing in the morning, melt 1L NGM in
  autoclave2Once melted, place in water bath set at 58oC, leave until cooled
  (-at least 2hours) normally left until the afternoon.3Set out the plates on bench before starting
  pouring process. 
    Put melted NGM media on stirrer
    4Using 1ml gilson add&nbsp; the Cholesterol,replace the lid and return
  to the bench to give a good stir to dispense all the cholesterol.5Add the KH2PO4 using sterile 50ml syringe and 0.22µm filter.6Stir for a few seconds until well mixed. Add
  Fungizone ensuring all is added to the NGM.7Dispense media using a peristaltic pump. Add
  4.20ml in 30mm plates, 9.00ml in 50mm plates, 4.50ml in 6 well plates and
  2.5ml in 12 well plates. When set stack upside down in plastic trays.
A1First thing in the morning, melt 1L NGM in
  autoclave2Once melted, place in water bath set at 58oC, leave until cooled
  (-at least 2hours) normally left until the afternoon.3Set out the plates on bench before starting
  pouring process. 
    Put melted NGM media on stirrer
    4Using 1ml gilson add&nbsp; the Cholesterol,replace the lid and return
  to the bench to give a good stir to dispense all the cholesterol.5Add the KH2PO4 using sterile 50ml syringe and 0.22µm filter.6Stir for a few seconds until well mixed. Add
  Fungizone ensuring all is added to the NGM.7Dispense media using a peristaltic pump. Add
  4.20ml in 30mm plates, 9.00ml in 50mm plates, 4.50ml in 6 well plates and
  2.5ml in 12 well plates. When set stack upside down in plastic trays.
TITLE: Cell cultures, transfection and treatments
AUTHORS: Federico Herrera

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Human glioblastoma U251 cells and rat brain glioma C6 cells were grown to confluence in Dulbecco´s Minimal Essential Medium (DMEM) supplemented with 10% (v/v )fetal bovine serum (FBS),1%(v/v)Penicillin-Streptomycin and 1% (v/v) L-glutamine, and maintained at 37ºC in a humidified atmosphere containing 5% CO</span><span style = "vertical-align:sub;">2</span><span>. Medium was changed every other day and cells were passed once a week by trypsinization. Briefly, cells were incubated with TrypLE Express dissociation reagent for 5 min at 37ºC for cell detachment, followed by a centrifugation step at 300 g for 5 min at room temperature and sub-cultured according to the desired dilution. For fluorescent microscopy and single-molecule tracking experiments, 3×10</span><span style = "vertical-align:super;">5 </span><span>cells were grown on 35 mm glass-bottom dishes (21 mm glass surface diameter, Ibidi GmbH, Gräfelfing, Germany). For flow cytometry, 3×10</span><span style = "vertical-align:super;">5</span><span> cells were seeded on 6-well plates (35 mm diameter, Corning, NY,USA). For Western blotting, 8×10</span><span style = "vertical-align:super;">5 </span><span>cells were seeded on 60 mm dishes (Orange Scientific, Braine-L’Alleud,Belgium). Twenty-four hours after seeding, cells were transiently transfected with 1 µg of the corresponding DNA constructs using jetPRIME transfection reagent (Polyplus transfection, Illkirch, France) in a proportion of 3:1 (µL of transfection reagent: µg of DNA).</span></div><div class = "text-block">All drugs and HaloTag ligands were diluted in DMSO (100% v/v). To test the neuroprotective activity of CNB-001 (0.1, 1 or 10 µM), cells were treated for 20 min before transfection and incubated for 24 h at 37ºC. For single-molecule experiments, 24 h after transfection, serum was removed for 2 h, and cells were treated with LIF (100 ng/ml), Nocodazole (10 µM) or Latrunculin B (10 µM) for 2 h at 37ºC in serum-free medium. Cells were then incubated with JF647 (100nM), JF549 (100 nM) or PA-JF549 (1nM) HaloTag ligands in serum-free medium for 20 min at 37ºC.Subsequently, cells were washed twice with sterile PBS. At the end of the final wash, the medium was changed to Leibovitz’s L-15 without phenol red.</div></div>

[STEPS]
TITLE: COVID-19: Hospital pharmacological treatment according to geographic region in Colombia
AUTHORS: Jorge         Machado Alba

[DESCRIPTION]
Introduction: The impact of COVID-19 prompted a race to find a treatment that would reduce its mortality. Most studies have not shown favorable results for many of these drugs, but they are still used.
Objective: To determine the differences and similarities in the hospital pharmacological management of patients with COVID-19 according to sex, age group, and geographical region of Colombia, 2020-2021.
Materials and methods: Descriptive cross-sectional study. We identified the prescription patterns of the medications given to patients diagnosed with COVID-19 treated in eight clinics in Colombia between March 6, 2020 and May 31, 2021. We performed a descriptive analysis of the sociodemographic, clinical, and pharmacological variables of the patients.
Results: A total of 8596 patients from 170 cities were identified, with a median age of 53.0 years and 53.3% of them men. A total of 24.3% required care in the intensive care unit, and 18.7% required invasive mechanical ventilation. The most commonly used drugs for the treatment of COVID-19 were systemic corticosteroids (63.6%), followed by colchicine (12.8%), azithromycin (8.9%), and ivermectin (6.4%). Corticosteroids, anticoagulants, colchicine, azithromycin, ivermectin, and hydroxychloroquine were prescribed more frequently in men, and their overall use increased with age. There were differences in prescriptions between geographic regions.
Conclusions: The majority of patients were managed with medications included in the management guidelines. There were differences between sexes, age groups, and geographical regions.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol provides method for KEGG Analysis, Gene Ontology Analysis, and Operational Protein Family Analysis. Based on the methods from the following publication: <br /><br />Hannigan, Geoffrey D., et al. "The Human Skin Double-Stranded DNA Virome: Topographical and Temporal Diversity, Genetic Enrichment, and Dynamic Associations with the Host Microbiome." <em>mBio</em> 6.5 (2015): e01578-15.

[BEFORE_START]
Perl scripts and other supplemental information available at:<br /><br /><a href="https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248" target="_blank">https://figshare.com/articles/The_Human_Skin_dsDNA_Virome_Topographical_and_Temporal_Diversity_Genetic_Enrichment_and_Dynamic_Associations_with_the_Host_Microbiome/1281248</a>

[GUIDELINES]
<ul>
<li>Output
<ul>
<li>For KEGG analysis:
<ul>
<li>KEGG_humann/04b-hit-keg-mpm-cop-nul-nve-nve-skinmet.txt</li>
<li>KEGG_humann/04b-hit-keg-mpm-cop-nul-nve-nve-virome.txt</li>
</ul>
</li>
<li>For GOEAST analysis
<ul>
<li>Step one input files in IntermediateOutput/GOEAST/
<ul>
<li>sebaceous_GO_ids_formatted.txt</li>
<li>moist_GO_ids_formatted.txt</li>
<li>rimoist_GO_ids_formatted.txt</li>
</ul>
</li>
<li>Step two input files in IntermediateOutput/GOEAST/
<ul>
<li>sebaceous_skinmet_uniprot_blastx_unique_probes.txt &amp; sebaceous_virome_uniprot_blastx_unique_probes.txt</li>
<li>moist_skinmet_uniprot_blastx_unique_probes.txt &amp; moist_virome_uniprot_blastx_unique_probes.txt</li>
<li>rimoist_skinmet_uniprot_blastx_unique_probes.txt &amp; rimoist_virome_uniprot_blastx_unique_probes.txt</li>
</ul>
</li>
<li>GOEAST output files (for visualization with Multi-GOEAST) in IntermediateOutput/GOEAST/output/
<ul>
<li>result_contigs-seb-skinmet.txt &amp; result_contigs-seb-virome.txt</li>
<li>result_contigs-moist-skinmet.txt &amp; result_contigs-moist-virome.txt</li>
<li>result_contigs-rimoist-skinmet.txt &amp; result_contigs-rimoist-virome.txt</li>
</ul>
</li>
</ul>
</li>
<li>For Operational Protein Family analysis
<ul>
<li>./protein_cluster_rel_abund_table/contig_otu_table_transposed.txt</li>
</ul>
</li>
</ul>
</li>
<li>R scripts: <a href="https://www.protocols.io/view/Script-R12-Functional-Analysis-ejibcke" target="_blank">R12</a> and <a href="https://www.protocols.io/view/Script-R13-OPF-and-AMG-Analysis-ejkbckw" target="_blank">R13</a></li>
</ul>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Protocol for using Mouse-FMRP antibody follow immunoprecipitation of FMRP using a rabbit antibody such as Abcam #17722

[GUIDELINES]
This protocol is still in progress, will be updated periodically.&nbsp;

[STEPS]
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TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Reagent List:</strong><br />
<ul>
<li>Cell Staining Buffer (BioLegend Cat. No. 420201)</li>
<li>Red Cell Lysis Buffer (BioLegend Cat. No. 420301)</li>
<li>7-AAD Viability Staining Solution (BioLegend Cat. No. 420403)</li>
<li>TruStain FcX™ (anti-CD16/32, BioLegend Cat. No. 101319)</li>
<li>Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat. No. 422301)</li>
</ul>
<br /><strong>References:<br /></strong>Current Protocols in Cytometry (John Wiley &amp; Sons, New York), Unit 6 Phenotypic Analysis.<strong><br /></strong>

[STEPS]
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TITLE: Transformation
AUTHORS: Ana Belem García González, Georgina Diego, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
A protocol for transforming E. coli cells is shown.

[STEPS]
SECTION: Transformation
1. Mix 50 µLof fresh competent cells with2 µLof genetic material (softly without pipetting) in a sterile 1.5 mL Eppendorf tube.

SECTION: Transformation
2. Incubate on ice for 30 min 

SECTION: Transformation
3. Apply thermal shock in a water bath at 42 °Cfor 30 s

SECTION: Transformation
4. Immediately, put on ice 

SECTION: Transformation
5. Add250 µL of SOC medium (tempered at 37 °C).

SECTION: Transformation
6. Incubate at 37 °C for60 min at  .  

SECTION: Transformation
7. Disseminate 50 µLin a selective medium and incubate 60 min at 37 °C
TITLE: VitalCare Nutrition Keto Gummies - World #1 Weight Loss Supplement
AUTHORS: G  A

[DESCRIPTION]
That means, when we run out - which we expect will occur in the following week or somewhere in the vicinity - you're in really bad shape. In this way, don't pass up a great opportunity! Request today and guarantee the best VitalCare Nutrition Keto! Your body will thank you!


[STEPS]
TITLE: Multi-dimensional potential factors associated with COVID-19 vaccine booster acceptance among the Bangladeshi people: a cross-sectional study
AUTHORS: dn.roy

[DESCRIPTION]
This protocol designed to assess the COVID-19 booster vaccine acceptance and identify the multi-dimensional potential factors influencing booster dose acceptance among the people in Bangladesh.Bangladeshi people of age 18 years and above were the participants for this analysis. To confirm the study reliability and rationalize the research objectives, we have collected the data from eight divisions in Bangladesh. An anonymous, multi-items, and closed-ended questionnaire was adopted from the theoretical analysis of the recent literature's on topic. A quantitative approach was used to analyze data from randomly selected people between August 2022─September 2022, which resulted in the participation of 607 respondents. As the response variable of the study, we measured willingness to uptake a booster vaccine and the responses were measured as a binary variable (1=Yes, 0=No). Binary logistic regression was employed to assess the study objectives. p<0.05 was considered the significant cut-point value. A face-to-face data collection denied the acceptance of incomplete survey response, thus, no missing data received. 


[STEPS]
TITLE: Sucrose lysis buffer
AUTHORS: Colleen Kellogg

[DESCRIPTION]
This protocol describes the preparation of sucrose lysis buffer to preserve DNA on sterivex filters. The protocol is part of the Hakai Institute's pipeline to analyze microbial and environmental DNA from seawater samples and is implemented as a standard procedure for ongoing sampling programs.

[BEFORE_START]
Read background information, MIOP and BePOP-OBON information under the "Guidelines" tab.

[STEPS]
SECTION: METHODS
9. Filter-sterilize and label bottle.
SECTION: METHODS
8. Top up water to 500 mL. (no need to pH this one!)
SECTION: METHODS
7. Add a stir bar and dissolve all powder.
SECTION: METHODS
6. Add milliQ water to about the 400 mL line.
SECTION: METHODS
5. Add 25 mL of 1 Molarity (M) Tris to the beaker.

Calculation of Tris or EDTA:
Use the equation C1V1 = C2V2

Eg for EDTA:
(0.5M)(X mL) = (0.04M)(500 mL)
Solve for X
((0.04M)(500 mL)/(0.5M)) = 40 mL of 0.5M EDTA
SECTION: METHODS
4. Add 40 mL of 0.5 Molarity (M) EDTA to the beaker.

Calculation of Tris or EDTA:
Use the equation C1V1 = C2V2

Eg for EDTA:
(0.5M)(X mL) = (0.04M)(500 mL)
Solve for X
((0.04M)(500 mL)/(0.5M)) = 40 mL of 0.5M EDTA
SECTION: METHODS
3. Weigh out and add appropriate amount of Sucrose calculated in step 1 and place in a bottle or beaker.
SECTION: METHODS
2. Calculate how much Sucrose powder you need (using the molecular weight on the bottle, MW or FW) for a 0.75 Molarity (M) solution of 500 mL.

Calculation of Sucrose:
 

Where Y is the molecular weight of the sucrose (MW or FW) from the bottle.
SECTION: METHODS
1. 

 

Equipment:
1 Molarity (M) Tris pH 8 (made previously or purchased as solution)
0.5 Molarity (M) EDTA pH 8 (made previously or purchased as solution)
Sucrose
MilliQ
500 mL bottle top filtration unit

Final concentrations of chemicals in SLB:
EDTA: 40 Molarity (M)
Tris: 50 Molarity (M)
Sucrose: 0.75 Molarity (M)
TITLE: Artificial Cerebrospinal Fluid III (ACSF.III)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Artificial Cerebrospinal Fluid III (ACSF.III) is used for applications including tissue bath solution during electrophysiological recording.</div><div class = "text-block"><span style = "font-weight:bold;">Note: </span><span>Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. </span></div></div>

[STEPS]
TITLE: Optical densitometry of tyrosine hydroxylase fibers
AUTHORS: mariangela.massarocenere

[DESCRIPTION]
Protocol for quantifying TH+ fibers in rat striatum and substantia nigra pars reticulata (SNpr)

[STEPS]
1. Acquire images with a light microscope of immunostained serial coronal sections covering the caudo-rostral of the brain regions (3sections/animal for striatum and SNpr)
2. Open ImageJ (RRID:SCR_003070,https://imagej.net/)
3. Calibrate grey mean values for standard optical density values 
4. Open 8-bit images in ImageJ, trace the region of interest (striatum, SNpr), and measure the optical density. Then, in the same image, trace the region of interest in a blank area (cortex for striatum, surrounding neuropil for SNpr ) and measure the optical density.
5. Calculate the optical density by subtracting the background optical density from the striatal optical density (if traced region of interest areas are the same) or by the formula: SNpr Integrated density-[(SNpr area) x (background optical density)]
TITLE: PBS with 0.1% Sodium Azide
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the method for preparing PBS with 0.1% Sodium Azide for storage and/or processing of fixed tissue.

Note: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
TITLE: Intracardial perfusion for electrophysiology in rat
AUTHORS: Alicia Avelar

[DESCRIPTION]
This protocol is to use for clearing blood from the brain and helping to maintain viable cells for electrophysiology experiments.  No PFA or any tools that have had contact with PFA should be used. This intracranial perfusion can help maintain viability of neurons in older animals and those who have undergone treatments that damage brain cells (for example: chronic methamphetamine and Parkinson's disease like treatments). 

[BEFORE_START]
Gather tools and set up the area and double check before beginning any surgical procedure. 

[GUIDELINES]
The rat should be unable to feel pain, but still breathing so the neurons are as healthy and in a physiological condition close to when the animal was alive when you attempt to do electrophysiology experiments later using the brain slices. 

[STEPS]
SECTION: Anesthesia
1. anesthetize rat with isoflurane anesthesia machine.
SECTION: Anesthesia
2. when rat is lying down immobile, doesn’t right it’s position when moved, and is breathing slowly put isoflurane nose cone on rat and test for pain with forceps toe pinch of both feet
SECTION: Surgery and intracranial perfusion
3. open rat thoracic cavity under sternum
SECTION: Surgery and intracranial perfusion
4. hold sternum back with forceps
SECTION: Surgery and intracranial perfusion
5. cut diaphragm
SECTION: Surgery and intracranial perfusion
6. cut sides of rib cage
SECTION: Surgery and intracranial perfusion
7. small cut to open right atria
SECTION: Surgery and intracranial perfusion
8. inject into heart ventricle ~ 120 mL ice cold artificial cerebral spinal fluid, then ~ 120 mL ice cold high sucrose solution
SECTION: Brain extraction
9. decapitate rat
SECTION: Brain extraction
10. carefully remove skull
SECTION: Brain extraction
11. gently free brain from skull, cut cranial nerves when necessary
SECTION: Brain extraction
12. let brain gently release from skull into ice cold high sucrose solution
SECTION: Cleanup
13. clean surgical tools carefully and let air dry
SECTION: Cleanup
14. sharps disposed of in sharps biohazard red box
SECTION: Cleanup
15. let blood dissolve in bleach in flask in hood to decontaminate
SECTION: Cleanup
16. dispose of other biohazard material in a double bagged biohazard red bag .add sticker label. dispose of in biohazard waste bins.
SECTION: Cleanup
17. clean surfaces in hood with ethanol and paper towels
SECTION: Cleanup
18. bring rat body in body bag with labeled tag to BSB 099 cold room
SECTION: Cleanup
19. bring cage and accessories with it to BSB dirty cage area for washing
TITLE: Semi-thin section analysis
AUTHORS: Sue Lin

[STEPS]
?. Double fixation: The specimen was first fixed with 2.5% glutaraldehyde in phosphate buffer (pH7.0) for more than 4 hours; washed three times in the phosphate buffer; then postfixed with 1% OsO4 in phosphate buffer (pH7.0) for 1 hour and washed three times in the phosphate buffer.
?. Dehydration: The specimen was first dehydrated by a graded series of ethanol (50%, 70%, 80%, 90%, 95% and 100%) for about 15 to 20 minutes at each step, transferred to absolute acetone for 20 minutes.
?. Infiltration: The specimen was placed in 1:1 mixture of absolute acetone and the final Spurr resin mixture for 1 hour at room temperature, then transferred to 1:3 mixture of absolute acetone and the final resin mixture for 3 hours and to final Spurr resin mixture for overnight.
?. Embedding and semi-thin sectioning: Specimen was placed in capsules contained embedding medium and heated at 70℃ for about 9 hours. Semi-thin sections (1 μm) were sliced under a LKB 11800 PYRAMITOME ultramicrotome (Stockholm, Sweden) and stained with 0.5% toluidine blue. Images of the anther cross-sections were obtained with a Leica DMLB fluorescence microscope (Leica Microsystems, Wetzlar, Germany).