eamag/protocols-with-steps huggingface dataset:
TITLE:     ELISA for quantification of IL-13 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. </div><div class = "text-block"><span>CD4+T cells (Th2), NKT cells and mast cells synthesize IL-13. It acts on monocytes, fibroblasts, epithelial cells and B cells. The IL-13 significant effects are B-cell growth and differentiation, stimulates isotype switching to IgE. It causes increased mucus production by epithelial cells, increased collagen synthesis by fibroblasts and inhibits pro-inflammatory cytokine production. Also, IL-13 works together with IL-4 in producing biologic effects associated with allergic inflammation and in defense against parasites. </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-13 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-13 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-13 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-human IL-13 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3',3',5',5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-13 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-13 standard dilutions and the human IL-13 sample concentration is determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: p2 11/5 
AUTHORS: Maria Guliakina

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">test</div></div>

[STEPS]
?. test
{"blocks":[{"key":"ccqb3","text":"","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Description of the automated tool, which was developed for quantifying the total and air-free volume of the maxillary sinus based on computed tomography images. The quantification tool seeks to standardize maxillary sinus volume measurements, thus allowing better comparisons and determinations of factors that influence maxillary sinus size. The automated tool utilized image processing techniques. </p>
<p> </p>
<p>Please contact PhD. Diana Rodrigues de Pina (drpina@fmb.unesp.br) for additional information regarding this protocol.</p>

[GUIDELINES]
<p>Please contact Dra. Diana Rodrigues de Pina (drpina@fmb.unesp.br) for additional information regarding this protocol.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Via Keto Apple Gummies Canada Reviews: Keto BHB ACV Formula in Canada UK, USA
AUTHORS: ViaKeto  Apple Gummies

[DESCRIPTION]
Via Keto Apple Gummies Canada Reviews: Keto BHB ACV Formula in Canada UK, USA

Visit the Official Website:- https://rvmac.net/viaketo-apple-gummies-canada/

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Suprarenal region of the abdominal aorta is the portion from the last intercostal artery to the right renal artery.In the Angiotensin II infused mouse model, abdominal aortic aneurysms are located in this region. Maximal outer width of the suprarenal aorta is a major parameter to define abdominal aortic aneurysms. Fifty % or more increase of this parameter, compared to control (saline infusion), is defined as an abdominal aortic aneurysm.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunostaining of iPSC-derived neurons
AUTHORS: Dan Dou, Erika L.F. Holzbaur

[DESCRIPTION]
Here, we fix, permeabilize, and stain human iPSC-derived neurons for the purpose of observing and quantifying somal and axonal abundance of proteins of interest. For preceding culture of neurons, see "Protocol: Culture and transfection of iPSC-derived neurons for live-imaging of axonal cargoes."

[STEPS]
1. Replace culture media in dish with 1 mL room temperature BrainPhys culture media (Stemcell Technologies)
2. Add 1 mL Bouin's solution (Sigma). Incubate at room temperature for 30 minutes.

3. Wash at least 5 times with PBS. Yellow color should be absent.
4. Permeabilize in ice-cold Optima methanol (Thermo Fisher) at -20 degrees C for 8 minutes.
5. Wash 3 times in PBS.
6. Block for one hour (5% goat serum and 1% BSA in PBS).
7. Incubate in primary antibody in blocking solution for 1 hour at room temperature.
8. Wash 3 times in PBS.
9. Incubate in secondary antibody in blocking solution for 1 hour at room temperature.
10. Wash 3 times in PBS.
11. Remove PBS and add 40 µL Prolong Gold (Thermo Fisher). Using forceps, apply coverglass.
TITLE: Total Starch Enzymatic Digestion 
AUTHORS: Lynn   Doran, Amanda P. De Souza

[DESCRIPTION]
Enzymatic digestion of total soluble starch to glucose in plant tissue extracts for preparation for quantification via the GOD-POD Method (NZYtech).

[BEFORE_START]
Extract and dry total starch pellet from plant tissue per Extraction of Non-Structural Carbohydrates (Total Soluble Sugars + Starch) in Plant Tissues.

[STEPS]
1. Prepare fresh daily 120 U/mL α-amylase in MOPS buffer. 1 mL per sample will be needed. Initial concentration of α-amylase is 3000 U/mL.  Use C1V1 = C2V2 to calculate the volume of α-amylase and MOPS buffer to use.
 

2. Prepare fresh daily 30 U/mL amyloglucosidase in acetate buffer. 1 mL per sample will be needed. Initial concentration of amyloglucosidase is 3260 U/mL.  Use C1V1 = C2V2 to calculate the volume of amyloglucosidase  and acetate buffer to use.
 

3. Heat the water bath to 75 °C.

4. Add 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.

5. Vortex to suspend all solids.  Flicking the tube may help dislodge the dried starch pellet.
6. Incubate for 30 min at 75 °C in the water bath.

7. Add another 500 µL of α-amylase in MOPS buffer (120 U/mL) to each sample tube.
8. Vortex to suspend all solids.
9. Incubate for 30 min at 75 °C in the water bath.

10. Cool down the water bath to 50 °C, tubes can be stored at room temp on the counter while water bath cools.
 

11. Add 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

12. Vortex to suspend all solids.
13. Incubate for 30 min at 50 °C in the water bath.

14. Add another 500 µL amyloglucosidase in acetate buffer (30 U/mL) to each sample tube.

15. Vortex to suspend all solids.
16. Incubate for 30 min at 50 °C in the water bath.

17. Place the tubes in ice to stop the reaction, until cool to the touch.
18. Proceed to total starch (as glucose) quantification by NZYtech GOD-POD method or store the samples at -20°C up to one month.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses <em>actual</em> reads from <a href="https://github.com/MicroB3-IS/osd-analysis" target="_blank">Ocean Sampling Day (2014)</a> and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure.

[BEFORE_START]
To run this protocol, users must first <a href="https://user.iplantcollaborative.org/register/" target="_blank">register</a> for Cyverse account. All data (both inputs and outputs) are available within Cyverse's data store at /iplant/home/shared/iVirus/ExampleData/<br /><br />
<ol>
<li>Download and install <a href="http://www.oracle.com/technetwork/java/javase/downloads/jdk8-downloads-2133151.html" target="_blank">Java JDK 8</a></li>
<li>Download and install <a href="http://www.cytoscape.org/download.php" target="_blank">Cytoscape 3.x</a></li>
</ol>

[GUIDELINES]
This is part of a larger protocol <em>Collection</em> that involves the end-to-end processing of raw viral metagenomic reads obtained from a sequencing facility to assembly and analysis using Apps (i.e. tools) developed by iVirus and implemented within the Cyverse cyberinfrastructure.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Second-derivative UV Spectroscopy
AUTHORS: Moriah Beck

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Second-derivative spectroscopy has proven to be an effective analytical tool because of its ability to resolve overlapping bands in the normal spectrum.</div><div class = "text-block">This protocol is used to determine the degree of tyrosine exposure in proteins using second-derivative UV spectroscopy.</div></div>

[STEPS]
?. [Protein Concentrations]
Determine protein concentrations either using absorption coefficients (Abs @ 280 nm) or using BCA, Bradford, etc. colorimetric assays.Proteins should be used at concentration where A280 ~ 0.2
?. [UV Scan]
Scan from 270-300 nm
?. [Obtain Second-derivative]
Copy UV scan data from instrument into a Prism file with columns for wavelength and absorbance values.
TITLE: Lentivirus Production and Astrocyte Transduction
AUTHORS: Shiyi Wang

[DESCRIPTION]
Lentivirus Production and Astrocyte Transduction

[STEPS]
1. 1. **Materials Required**
1.1. - pLKO.1 shRNA Puro targeting plasmid (for astrocyte transduction)
1.2. - Envelope plasmid (VSVG)
1.3. - Packaging plasmid (dR8.91)
1.4. - HEK293T cells
1.5. - X-tremeGENE transfection reagent (Roche)
1.6. - Astrocyte growth media (AGM)
2. - Puromycin (for selection)
2.1. 2. **Transfection of HEK293T Cells**
2.2. - Plate HEK293T cells in appropriate culture vessels (e.g., T75 flask) to achieve 70-80% confluence on the day of transfection.
2.3. - Prepare transfection mix per flask:
2.4. - Combine 2 μg pLKO.1 shRNA Puro plasmid, 1.5 μg VSVG plasmid, and 1.5 μg dR8.91 plasmid in Opti-MEM.
2.5. - Add X-tremeGENE transfection reagent according to the manufacturer’s instructions.
2.6. - Incubate transfection mix at room temperature for 20 minutes.
2.7. - Add transfection mix dropwise to cells and gently swirl flask to mix. Incubate at 37°C with 5% CO2.
3. 3. **Collection of Lentivirus**
3.1. - Replace media with fresh AGM 24 hours post-transfection to enhance virus production.
3.2. - Collect lentivirus-containing media on days 2 and 3 post-transfection.
3.3. - Filter collected media through a 0.45 μm syringe filter to remove cell debris.
3.4. - Store lentivirus aliquots at -80°C for future use.
4. 4. **Transduction of Rat Primary Astrocytes**
4.1. - **Day 7 In Vitro (DIV 7)**
4.2. - Plate rat primary astrocytes in 6-well dishes at a density suitable for transduction experiments (e.g., 2 ml AGM per well).
4.3. - **Day 8 In Vitro (DIV 8)**
4.4. - Remove 1 ml of AGM from each well and replace with 500 μl fresh AGM + 500 μl lentivirus-containing media.
4.5. - Add 1 μg/ml polybrene to enhance transduction efficiency.
4.6. - **Day 10-15 In Vitro (DIV 10-15)**
4.7. - Treat transduced astrocytes with puromycin (1 μg/ml) to select for cells expressing the shRNA construct.
4.8. - **Day 15 In Vitro (DIV 15)**
4.9. - Lysate astrocytes to extract proteins for Western blot analysis to assess knockdown efficiency.
5. Notes:
5.1. - Handle lentiviruses in a biosafety level 2 (BSL-2) laboratory following institutional guidelines.
5.2. - Perform all steps involving lentivirus production under sterile conditions to prevent contamination.
5.3. - Optimize lentiviral titration to achieve desired transduction efficiency in astrocyte cultures.
5.4. - Ensure proper disposal of materials used for lentivirus production according to institutional biohazard waste disposal protocols.
TITLE: Depression Detection Algorithm
AUTHORS: Umme Marzia  Haque

[DESCRIPTION]
The study has used data from YMM. The Yes/No variables that had a low correlation with target variable have been removed. To extract the most relevant features , the high correlated variables with the target variable , the Boruta method was used in conjunction with a Random Forest( RF) Classifier. To select suitable supervised learning models, the Tree-based Pipeline Optimization Tool To select suitable supervised learning models, the Tree-based Pipeline Optimization Tool (TPOTclassifier) has been employed. RF, XGBoost (XGB), Decision Tree (DT), and Gaussian Naive Bayes (GaussianNB) have been employed in the depression identification step.has been employed. RF, XGBoost (XGB), Decision Tree (DT), and Gaussian Naive Bayes (GaussianNB) were employed in the depression identification step.

[STEPS]
TITLE: Mouse Stellate- Intracellular recording
AUTHORS: John Tompkins, Mike Andresen

[STEPS]
?. See mouse stellate iosolation protocol
?. Isolated mouse stellate pinned to clean Sylgard perti dish
?. Continuously superfuse isolated stellate (6–7 ml/min) with PSS (32–35°C).
?. Impale individual neurons using microelectrodes filled with either 2M KCl (60–120 MΩ) or 2M KCl + 2% Neurobiotin (80-160 MΩ; Vector Labs).
?. Record intracellular membrane voltage using a Multiclamp 700B amplifier coupled with Digidata 1550B data acquisition system and pCLAMP 10 software (Molecular Devices, Sunnyvale, CA).
?. Characterize responsiveness of membrane potential to membrane physiology. Depolarizing current steps (0.1–0.5 nA amplitude, 500-ms or 1-s duration) are used to assess neuronal excitability.Cells are classified as either phasic (5 spikes continuously) based on the number of APs elicited by the intracellular current. Hyperpolarizing current steps (500 ms) of increasing amplitude are used to test for rectification in the current-induced hyperpolarization.Action potential (AP) amplitude and duration are measured from spontaneous or nerve evoked APs.After-hyperpolarization amplitude and duration were measured from brief intracellular current injections (100-500pA, 5ms), spontaneous APs, or nerve evoked APs.
?. Graded stimulus shocks (100 µs) delivered from the concentric bipolar electrodes to identified nerves in 50-100μA steps, from 800uA to 0μA were used to generate a stimulus recruitment curves (AMPI Master 8 and IsoFlex optical Isolation unit). 5-20 stimuli were delivered at each stimulus intensity, with an interval of 3 sec between stimuli. Analysis of synaptic events focused on EPSP latency and jitter (SD of latency) as indices of conduction, path and release.
TITLE: Calculating mitochondrial protein solubility
AUTHORS: Louise Uoselis

[DESCRIPTION]
Protocol for generating solubility calculations for mitochondrial proteins from starting 'soluble' and 'insoluble' fraction mass spectrometry data, using MaxQuant and Perseus software pipelines.

[STEPS]
1. Process raw instrument files using MaxQuant (protocol performed with v1.6.17) with the Andromeda search engine, searching against the Uniprot human data base containing reviewed and canonical isoform variants in the FASTA format, with recombinant Ag85A sequence added as a custom entry to the human database
1.1. Set the LC-MS run to "Reporter ion MS"
1.3. Set Trypsin/P cleavage specificity to a maximum of 2 missed cleavages 
1.2.  Set TMT11-plex labels as isobaric labels with a reporter ion mass tolerance of 0.003 Da
1.4. Set oxidation of methionine and N-terminal acetylation as variable modifications, and carbamidomethylation of cysteine as a fixed modification
1.5. Enable the 'Match between runs' option, with an FDR o f`% and mass bin size of 0.0065 Da
1.6. Set minimum unique and razor peptides to 1, and label min ratio count to 2
1.7. Run the MaxQuant analysis
2. Extract the proteinGroups.txt table, and normalise the data following the protocols outlined by:

D. L. Plubell, P. A. Wilmarth,
Y. Zhao, A. M. Fenton, J. Minnier, A. P. Reddy, J. Klimek, X. Yang, L. L.
David, N. Pamir, Extended Multiplexing of Tandem Mass Tags (TMT) Labeling
Reveals Age and High Fat Diet Specific Proteome Changes in Mouse Epididymal
Adipose Tissue. Mol Cell Proteomics 16, 873-890 (2017).

3. Average the internal spiked control (Ag85A) intensities across each reporter channel, and use this value to generate a scaling factor for each channel to normalise reporter ion intensities for each protein to the relative starting intensities in each sample at the time of Ag85A addition
4. Impute the data into Perseus and remove 'only identified by site', 'reverse', and 'potential contamination' identifications
5. Filter the clean data according to mitochondrial associated proteins, using a database such as the database found in:

I. Kuznetsova, A. Lugmayr, O. Rackham, A. Filipovska, OmicsVolcano: Software for intuitive visualization and interactive exploration of high-throughput biological data. STAR Protocols 2, 100279 (2021).

6. Generate computationally derived 'total' fraction intensities by addition of the 'soluble' and 'insoluble' total fraction intensities
7. Calculate the percentage of insoluble protein for each protein by dividing the insoluble protein intensity by the calculated total protein intensity and multiplying the outputted value by 100
TITLE: Reverse transcription of complementary DNA
AUTHORS: Madeline M Glennon, Austin Skinner, Mara Krutsinger, Marino Resendiz

[STEPS]
?. [Reverse Transcription using AMV RT]
Obtain an RNase -free microcentrifuge tube Add the following components in the described order:-Water -10 X Buffer -5'-P32 Radiolabeled DNA -RNA
400 µl
15 µl
[50 mM Tris-acetate, 75 mM Potassium Acetate, 8 mM Magnesium Acetate, 10 mM DTT (pH 8.3)]
[< 1 pmol]
[app. 2 pmol]
?. [Reverse Transcription using AMV RT]
Place the tube in a heat block at   and turn off the heat block to allow for slow cooling.
90 °C
?. [Reverse Transcription using AMV RT]
Remove the tube upon reaching room temperature and centrifuge for  using a microcentrifuge
?. [Reverse Transcription using AMV RT]
The solution is then transferred to a separate microcentrifuge tube
3 µl
?. [Reverse Transcription using AMV RT]
Add the desired dNTP solution
1 µl
?. [Reverse Transcription using AMV RT]
Add the reverse transcriptase solution  .  The reverse transcriptase solution consits of RT  dissolved in water  .  This enzyme was purchased from NEB.
2 µl
2 µl
58 µl
?. [Reverse Transcription using AMV RT]
Place the solution in an incubator at   for
37 °C
?. [Reverse Transcription using AMV RT]
Remove from water bath and add urea
7 µl
?. [Reverse Transcription using AMV RT]
Centrifuge for  using a microcentrifuge
?. [Reverse Transcription using AMV RT]
Place the tube in a heat block at   for
90 °C
?. [Reverse Transcription using AMV RT]
Centrifuge for  using a microcentrifuge while allowing to cool to rt
?. [Reverse Transcription using AMV RT]
Withdraw the solution   and load onto a denaturing 20 % PAGE
[This typically accounts for app 4000 cpm]
?. [Reverse Transcription using AMV RT]
On a separate well, add a marker composed of xylene cyanol and bromophenol blue in 90% formamide buffer
?. [Reverse Transcription using AMV RT]
Turn on the power supply and allow to run until the bromophenol blue marker is 3/4 down the gel.Settings will vary depending on the gel size and thicknes.
?. [Reverse Transcription using AMV RT]
Remove the gel from the gel stand and remove one glass slide.
?. [Reverse Transcription using AMV RT]
Develop using an autoradiography cassette
?. [Reverse Transcription using AMV RT]
Visualize and quantify the gel using a Molecular Dynamics Phosphorimager 840
?. [Reverse Transcription using MMLV RT]
Repeat all steps above, except with the amount of reverse transcriptase added. Add the reverse transcriptase solution  .  The reverse transcriptase solution consits of RT  dissolved in water  .  This enzyme was purchased from NEB M0253L at 200,000 U/mL
2 µl
1 µl
10 µl
?. [Reverse Transcription using HIV RT]
Repeat all steps above, except with the amount of reverse transcriptase added. Add the reverse transcriptase solution  .  The reverse transcriptase solution consits of RT  dissolved in water  ; followed by a second 1:1 dilution in water.  This enzyme was purchased from Worthtington LS05003 at 27.5 units/uL
2 µl
0.75 µl
1.5 mL
TITLE: Diaetoxil Kapseln Avis- FRance Prix, Medical Fake Avantage
AUTHORS: health  

[DESCRIPTION]
Diaetoxil Avis - Diaetoxil est l'un des suppléments de perte de poids les plus fiables. Il peut être utilisé par n'importe qui, quel que soit son âge, car il est extrêmement sûr à utiliser.

[STEPS]
1. Diaetoxil Avis - Diaetoxil est l'un des suppléments de perte de poids les plus fiables. Il peut être utilisé par n'importe qui, quel que soit son âge, car il est extrêmement sûr à utiliser. Diaetoxil Ingrédients, Résultats Une alimentation propre se concentre sur la consommation d'aliments entiers peu transformés et aussi proches que possible de leur forme naturelle. Adopter un régime alimentaire sain peut être un moyen simple et efficace de perdre du poids et d'améliorer votre état de santé général. De plus, une alimentation propre signifie moins dépendre des produits transformés achetés en magasin et préparer plus de repas à la maison, ce qui peut vous faire économiser de l'argent. Voici 24 conseils pour une alimentation propre qui peuvent vous aider à être en meilleure santé et plus énergique. De nombreuses habitudes de vie simples peuvent aide à la perte de poids. Certains n'ont rien à voir avec un régime alimentaire conventionnel ou des plans d'exercice. Vous pouvez utiliser des assiettes plus petites, manger plus lentement, boire de l'eau et éviter de manger devant la télévision ou l'ordinateur. Diaétoxil Donner la priorité aux aliments amaigrissants pour femmes riches en protéines et en fibres visqueuses peut également aider. Cependant, il est probablement préférable de ne pas essayer toutes ces choses à la fois. Expérimentez une technique pendant un certain temps, et quand cela fonctionne bien pour vous, essayez-en une autre. Quelques changements simples peuvent avoir un impact considérable sur votre poids à long terme. Site officiel : https://ipsnews.net/business/2022/06/29/diaetoxil-gelules-avis-france-2022-pharmercie-prix-perte-de-poids-capsules-arnaque-or-acheter/
TITLE: Defrost a C. elegans strain from the -80 C freezer
AUTHORS: Cristian Riccio

[STEPS]
?. Take the frozen C. elegans tube out of the -80 C freezer.
?. Put the tube in a foam floater.
?. Put the foam floater on the surface of lukewarm water in a beaker.
?. When the content of the tube has defrosted (should take only a few minutes), pour the contents onto an NGM plate seeded with bacteria.
?. Put the plate in a plastic box and put the box into an incubator, for example a 20 C incubator.
?. The following day, check that some worms are alive. I would recommend having at least 60 living pre-reprodutive or reproductive worms on the plate.