eamag/protocols-with-steps huggingface dataset:
TITLE: Immunofluorescence on paraffin sections
AUTHORS: miquel.vila

[DESCRIPTION]
Immunofluorescence protocol on paraffin-embedded rat brain sections 

[STEPS]
SECTION: 1. Deparaffinization and hydratation :
1. Put the slides 30 min in the incubator at 60ºC
SECTION: 1. Deparaffinization and hydratation :
2. Wash 3x3 min in xylene
SECTION: 1. Deparaffinization and hydratation :
3. Wash 1x10 min in ethanol 100%
SECTION: 1. Deparaffinization and hydratation :
4. Wash 1x10 min in ethanol 95%
SECTION: 1. Deparaffinization and hydratation :
5. Wash 1x5 min in ethanol 70%
SECTION: 1. Deparaffinization and hydratation :
6. Wash 2x5 min in PBS 1X
 

SECTION: Antigen retrieval
7. Add 200 mL/section of citrate buffer 10mM, pH 6
SECTION: Antigen retrieval
7.1. Put sections in a boiling water bath for 20 min
SECTION: Antigen retrieval
8. Let sections cool down for 20 min at room temperature (RT)
SECTION: Washing
9. Wash 3x5 min in PBS 1X-Triton (0.5%)
SECTION: Blocking
10. Put wet paper in a black box for immunohistochemistry
SECTION: Blocking
11. Gently dry and circle sections with the hydrophobic pen ImmEdge Vector H 4000 without touching the tissue 
SECTION: Blocking
12. Add 200 uL/section of blocking solution (5% NGS (in PBS 1X) (200uL/section) +  0.1% Triton (in PBS 1X)) 1h at RT
SECTION: 1ary antibody
13. Gently wipe off the water ofthe slides
SECTION: 1ary antibody
14. Put 200ul/section of 1ary antibody in 2% NGS + 0.1% Triton overnight at 4ºC (cold room)
SECTION: Washing
15. Wash 3x5min in PBS 1X-Triton (0.5%)
SECTION: 2ary antibody
16. Gently wipe off the water ofthe slides
SECTION: 2ary antibody
17. Put 200ul/section of 2ary antibody in 2% NGS + 0.1% Triton + DAPI for 1 h at RT
 

SECTION: Washing
18. Wash 3x5min in PBS 1X 
SECTION: Mounting
19. Mount sections with fluorescent mounting medium. Put the coverslip (washed with ethanol previously) on the slide. Remove bubbles
SECTION: Storage
20. Let dry the slides on the tray in the hood for 5 minutes and store at 4ºC asap
TITLE: Example of two-path protocol
AUTHORS: Cleyde Helena

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is an example of a protocol that have a common first steps, then forks into two different ones.</div></div>

[STEPS]
?. [Amount]
Add of Solution A into a beacker
10 µl
?. [Concentration]
Prepare of 0.9% NaClAdd of PBS
100 ml
20 ml
[(adjust pH to 7)]
?. [Incubation]
Mix Steps 1 and 2 at  for
?. [Incubation]
Incubate an extra
?. [Incubation]
Let it cool down and store in a freezer
?. [Incubation]
proceed with the experiment by injecting the solution on an Agilent 7890B GC system
TITLE: Polyelectrolyte-chain-at-equilibrium
AUTHORS: Mike Edwards

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>By means of the density functional theory framework (DFT) as well as the  molecular dynamic simulations (MD), a polyelectrolyte chain (PE) in the  good solvent conditions at thermal equilibrium is studied. The strength  of the electrostatic interactions is varied by the Bjerrum length of  the solvent. It turns out that average extension of a PE scales with the  degree of polymerization, very much similar to a neutral polymer chain  in good solvent. Remarkably, the difference between a PE and a neutral  chain appears to be solely in the correlations among monomers which are  stored in the Virial coefficients. Interestingly, upon increasing the  Bjerrum length of solvent, the chain shrinks. This outcome is confirmed  by the DFT framework as well as the MD simulations. The significance of this study is that it strongly criticizes the idea (already mentioned in T. Kreer, </span><span style = "font-style:italic;">Soft Matter</span><span>, </span><span style = "font-weight:bold;">12</span><span>,  3479 (2016)) that the PEs behave similar to a neutral ideal chain. This  study could be useful in our understanding of biopolymers.</span></div></div>

[STEPS]
TITLE:     ELISA for quantification of IL-20 in human serum or plasma.
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>Immune cells and activated epithelial cells secrete IL-20. It acts on epithelial cells. It plays a vital role in the cellular communication between epithelial cells and the immune system under inflammatory conditions.</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-20 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C.
?. Add 50 µl of human serum. Human IL-20 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-20 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-20 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-20 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-20 standard dilutions and the human IL-20 sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: IGVF Liver nuclei isolation protocol
AUTHORS: Elisabeth Rebboah

[DESCRIPTION]
This protocol describes isolation of nuclei from 10 week old whole mouse livers from 8 founder strains (B6J, AJ, 129S1J, NZOJ, WSBJ, NODJ, PWKJ, and CASTJ), preparation of a single nucleus suspension, and fixation for single nucleus RNA-seq using Parse Biosciences and SHARE-seq using part of the Broad Institute protocol. We process 1 rep from each strain per day; e.g. female rep 1 across all 8 strains. The main products we use are Parse Biosciences Nuclei Fixation Kit (v2) and Miltenyi Biotec’s gentleMACS Octo Dissociator with accessories. This protocol takes about 3.5 hours from start to finish.

[STEPS]
1.
TITLE: HuBMAP | GE/ University of Washington  Cell DIVE™ Modality Overview
AUTHORS: Liz  McDonough

[DESCRIPTION]
This is an overview of all protocols currently in use for the GE/University of Washington Cell DIVE collaboration for the Human BioMolecular Atlas Program (HuBMAP). It includes links to each of the individual protocols that make up this project workflow.

[STEPS]
2. Prepare paraffin blocks and FFPE sections from tissue samples.

HuBMAP Tissue Preservation Protocol
3. Deparaffinize and rehydrate slides.

Cell DIVE™ Platform | Slide Clearing and Antigen Retrieval
4. Characterize antibodies (primary/secondary, direct conjugates, and zenon labelled antibodies) and determine any antigen effects from the Cell DIVE dye inactivation process.

Cell DIVE™ Platform | Antibody Characterization for Multiplexing 
Cell DIVE™ Platform | Antibody Staining & Imaging

5. Prepare direct conjugates for study.

Cell DIVE™ Platform | Antibody Purification Chemistry
Cell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation
6. Perform Cell DIVE™ multiplexed data acquisition on the final cohort. 

 

1. Protocol for Tissue Collection from Organ Procurement Organization
TITLE: Species delimitation of Ophiothrix using bPTP
AUTHORS: Renata Alitto, Michela Borges, Letícia Dias de Oliveira, Karin Regina Seger, Luciana Bolsoni Lourenço

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This test adds Bayesian support (BS) values to the nodes of the input tree and delimit species based on the Phylogenetic Species Concept. Higher BS values indicate all descendants from the node are more likely to be from one species</div></div>

[STEPS]
?. [Phylogenetic tree]
The phylogenetic tree generated by the Bayesian Inference was submitted to the Bayesian Poisson Tree Processes - bPTP [56] to test species boundaries.
?. [Analyses]
The analyses were conducted on the web server (available at http://species.h-its.org/ptp/). The parameters for the run were 500000 MCMC generations, thinning of 100, and Burn-in of 0.25.
TITLE: Data and script to reproduce the analysis in &#39;Changes in methodological study characteristics in psychology between 2010-2021&#39;
AUTHORS: Ingmar Böschen

[DESCRIPTION]
This protocol contains the R script and data files to reproduce the analysis presented in: Böschen, I. (2022).Changes in methodological study characteristics in psychology between 2010-2021. In review.

[BEFORE_START]
Install a current R version (https://www.r-project.org/) and download the four data files and R script.

[STEPS]
1. Download the four data files and the R script provided at:
https://www.protocols.io/workspaces/methodological-changes-in-psychology
2. Open the script in R, set the working directory and run the analysis.
TITLE: RNA Isolation from Plant Tissue Protocol 5: pBIOZOL Method
AUTHORS: 

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Implemented by: Beijing Genomics Institute</div><div class = "text-block"><span>This protocol is part of a collection of eighteen protocols used to isolate total RNA from plant tissue. (RNA Isolation from Plant Tissue Collection: </span><a href="https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.protocols.io/view/rna-isolation-from-plant-tissue-439gyr6</span></a><span>) </span></div></div>

[STEPS]
?. Grind tissue to a powder in liquid nitrogen.
?. Add  of cold () pBIOZOL Reagent for up to  of frozen, ground tissue.
1.3 ml
4 °C
100 mg
?. Incubate the tube for  at .
20 °C
Lay the tube down horizontally to maximize surface area during RNA extraction.
?. Centrifuge for  at .
?. Add  of  NaCl to the extract.
100 µl
?. Add  of chloroform.
300 µl
?. Centrifuge the sample at  for  at  to separate the phases.
4 °C
?. Add to the aqueous phase an equal volume of isopropyl alcohol.
?. Centrifuge the sample at  for  at .
4 °C
?. Decant the supernatant, taking care not to lose the pellet.
?. Centrifuge at  for  at .
0 Room temperature
?. Add – RNase-free water to dissolve the RNA.
10 µl
30 µl
?. Mix by briefly vortexing or flicking the bottom of the tube until the sample is thoroughly suspended.
?. Transfer the supernatant to an RNase-free tube.
?. Tap tube to mix.
?. Mix thoroughly by inversion.
?. Transfer the top aqueous phase to an RNase-free tube.
?. Mix.
?. Let stand at  for .
20 °C
?. Add  of chilled 75 % ethanol to the pellet.
1 ml
Pellet may be difficult to see.
?. Decant the liquid carefully, taking care not to lose the pellet.
?. Briefly centrifuge to collect the residual liquid and remove it with a pipette.
?. Pipette the water up and down over the pellet to dissolve the RNA.
If any cloudiness is observed, centrifuge the solution at   for   at   and transfer the supernatant to a fresh tube.
TITLE: Plasmid Miniprep for 2022
AUTHORS: Team Fudan iGEM

[DESCRIPTION]
Plasmid extraction and purification, based on Vazyme Mini-prep Kit manual.

[STEPS]
1. Pellet bacteria culture in a 1.6mL centrifugation tube at 13,000rpm for 1 minute. Decant supernatant. The pellet in the tube can be stored in -20 freezer if time not enough for the following steps.
2. Add 250uL of resuspension solution (P1 with RNase) and vortex. After adding RNase into the P1, P1 is stored in 4 degree refrigerator.
3. Add 250uL of lysis solution (P2) and invert gently 4-6 times. P2 contains NaOH, and must keep tightly closed.
4. Add 350uL of neutralization solution (P3) and invert gently 4-6 times. Do not vortex, which will break bacteria genomic DNA and genomic DNA fragments will contaminate purified plasmids.
6. Take the clear supernatant, without any white protein cloud. Bind the DNA to the column by decanting.
7. Centrifuge at 13,300 rpm for 1 minute.
1 min
8. Wash the column with 600uL of wash solution (PW2) and centrifuge for 1 minute. Remind adding pure ethanol into PW2 as indicated on the bottle.
1 min
9. Discard the flow-through, wash again with 600uL of PW2, and centrifuge for 1 minute. Discard the flow-through and centrifuge the column again for 2 minutes.
3 min
10. Keep the cap open and wait until no ethanol smell. Move the column into a new centrifugation tube. Elute the purified DNA with 50uL of elution buffer (TE; 10 mM Tris-Cl, 1 mM EDTA). Then, centrifuge for 1 minutes. Collect the flow-through, which is the plasmid DNA. Because pET28 based is low-copy plasmid, pre-warm TE to 50 degree helps the elution.
1 min
5. Spin at 13,000rpm for 10 minutes, at 4 dgree.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Learn the basic commands to navigate in a command-line terminal.</p>

[BEFORE_START]
<p><strong>For Windows users :</strong></p>
<p>You will need to install a shell, I personnaly recommand Cygwin. To install Cygwin, go to <a href="https://www.cygwin.com/install.html" target="_blank" rel="noopener noreferrer">this page and follow the installation instructions for windows</a>.</p>
<p> </p>
<p><strong>For MacOS and Linux users :</strong></p>
<p>A shell is provided in your OS, no need to install anything. </p>
<p>MacOS users can however choose to use iTerm2 instead of the built in shell. To download and install iTerm2, go to <a href="https://iterm2.com/downloads.html" target="_blank" rel="noopener noreferrer">this page and follow the instructions</a>.</p>

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

[DESCRIPTION]
<p>Milliplex Cytokine/Chemokine 7-plex manufacturer's protocol</p>

[STEPS]
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?. 
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?. 
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?. 
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TITLE: Plate assay for identification of salt stress induced changes in Arabidopsis thaliana's Root System Architecture
AUTHORS: Magdalena Julkowska

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocols is a fairly simple method to study the developmental changes in Root System Architecture of Arabidopsis and other small plants. By germinating the plants on control medium, the </div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Infiltration of leaves with various herbicides, targetting photosynthetic electron transport, were utilized to mimic changes in redox state (otherwise caused by changes in light irradiance) of photosynthetic electron transport components and study the response of APX2. </p>
<p> </p>
<p>This was successfully  utilized in the following publication: </p>
<p>Jung, H., Crisp, P.A., Estavillo, G.M., Cole, B., Hong, F., Mockler, T.C., Pogson, B.J., and Chory, J. (2013). Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light. Proc. Natl. Acad. Sci. 110: 14474–14479.</p>

[BEFORE_START]
<p>Make sure healthy, low-moderate light acclimated plants are growing (of desired genotypes) with well expanded leaves ( leaf # 4 - 7 are generally good; ~3 - 4 week old plants w/ 12-hr day/night cycle).</p>

[GUIDELINES]
<p>Plants are ideally low/moderate light acclimated (&lt; 150 µ mol m<sup>-2</sup> s<sup>-1</sup>) that should allow induction of redox change (e.g. oxidation vs reduction) of targetted photosynthetic electron transport components (e.g. cytochrome bf complex, PQ pool).</p>

[STEPS]
?. 
?. 
?. 
?. 
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TITLE: Senescence Cocktail
AUTHORS: Sakthikumar Mathivanan

[DESCRIPTION]
Modeling age-related neurodegenerative disorders with human stem cells is difficult due to the embryonic nature of stem cell derived neurons. We developed a chemical cocktail to induce senescence of iPSC-derived neurons to address this challenge. We first screened small molecules that induce embryonic fibroblasts to exhibit features characteristic of aged fibroblasts. We then optimized a cocktail of small molecules that induced senescence in fibroblasts and cortical neurons without causing DNA damage. The utility of the “senescence cocktail” was validated in motor neurons derived from ALS patient iPSCs which exhibited protein aggregation and axonal degeneration substantially earlier than those without cocktail treatment. Our “senescence cocktail” will likely enhance the manifestation of disease-related phenotypes in neurons derived from iPSCs, enabling the generation of reliable drug discovery platforms.

[STEPS]
TITLE: Highfield Diagnostics COVID19 LFA Protocol
AUTHORS: peijun he

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Highfield Diagnositc`s has developed a COVID-19 rapid diagnostic test (RDT), which is a lateral flow immunochromatographic assay (LFA), that detects nucleoproteinin nasal swab specimens.The test provides a simple colorimetric readout of results in less than 30 minutes (including sample preparation time) at a cost of <$5 per test, with no special reader or additional equipment. This scalable and accessible solution puts simple and frequent testing capacity, without the need for skilled personnel, into the hands of billions across the world. </div></div>

[STEPS]
TITLE: AAV Craniotomy
AUTHORS: Santiago Unda, Roberta Marongiu, Mihaela Stavarache, Michael G. Kaplitt

[DESCRIPTION]
This protocol is for injection of AAV into a mouse brain using a stereotactic frame and Hamilton syringe.

[GUIDELINES]
Note that sterile technique must be followed during this procedure to avoid health complications.

[STEPS]
SECTION: Anesthesia
1. Weigh mice.
SECTION: Anesthesia
2. Administer intraperitoneal mixture of ketamine (ButlerAnimal Health Supply) and xylazine (Lloyd Laboratories) at concentrations of 110 and 10 mg/kg of body weight, respectively.
SECTION: Anesthesia
3. Confirm induction of anesthesia.
SECTION: Injection
4. Place mouse into stereotactic frame (David Kopf Instruments) and place eye lubricant.
SECTION: Post-procedure
17. Give meloxicam 2mg/kg subcutaneously.
SECTION: Injection
5. Remove fur from skull area using either electric shaver or depilatory cream.
SECTION: Injection
6. Sterilize skull area and then cut open with scalpel.
SECTION: Injection
7. Adjust brain so that it is level. Locate bregma and zero X and Y axes on stereotaxic frame.
SECTION: Injection
8. Move to injection coordinates and drill small hole into the skull.
SECTION: Injection
9. Zero the Z axis on the stereotaxic frame.
SECTION: Injection
11. Using a 10 μL stereotactic syringe with a 33 G needle attached to a micro-infusion pump (World Precision Instruments), withdraw the AAV solution.  
SECTION: Injection
14. To prevent reflux, after each infusion, leave the injection needle in place for 5 min, withdraw a short distance (0.3 - 0.5 mm), and then leave in the new position for an additional 2 min before complete removal of the needle.
SECTION: Injection
12. With the loaded syringe, navigate the needle to the desired X, Y, Z coordinates.
SECTION: Injection
13. Infuse the brain with AAV at a flow rate of 0.1 - 0.4 μL/min. 
SECTION: Injection
15. Close skin with non-absorbable sutures.
SECTION: AAV Preparation
16. Inject a total of 2x1011 genomic particles/mL of vector is injected into the appropriate coordinates. 
SECTION: Injection
10. Prepare AAV solution by diluting AAV in PBS with 500mM Mg2/Cl to a final dilution of total of 2x1011 genomic particles/mL.
SECTION: Post-procedure
18. Perform health checks every day for the first 72 hrs post-injection.
SECTION: Post-procedure
19. Remove sutures 15 days post-injection.
TITLE: Swallowing Study Using Water-soluble Contrast Agents Increases Aspiration Sensitivity and Antedates Oral Feeding without Respiratory and Drug Complications: A Prospective, Observational, Case-control Trial
AUTHORS: Chang Ho  Hwang

[STEPS]
SECTION: Materials
2. 


1. Based on their medical conditions and aspiration risk, patients undergoing the swallowing study were assigned to iohexol 350 (Omnipaque® [osmolarity 541 mOsm/L, viscosity 10.4 centipoise at 37°C, specific gravity of 1.406], GE Healthcare, USA)19 or barium sulfate (BaSO4 40% [osmolarity 233 mOsm/L, viscosity 2.3 centipoise at 25°C]) as the CA. 

Patients with a history of iodine allergy underwent MBSS. All patients were evaluated by a physiatrist with an intra-rater reliability of oropharyngeal swallowing efficiency of 0.87. The demographic and clinical characteristics of all patients were collected, including underlying diseases, performance of tracheostomy, feeding methods, radiologic findings before and after the swallowing study, total hospital stay, and days to discharge. Results suggesting pneumonitis, pulmonary edema, allergic responses, or chemotoxicity-related laboratory/vital findings were collected over 1 week after the evaluations. Radiologic change was interpreted by six independent radiologists.

If present, Levin tubeswere removed > 4 hours before the evaluations and oxygen was provided, as requested. Based on the penetration-aspiration scale, aspiration was defined as CA passing below the vocal folds. Patients were asked to sit in a wheel-chair with neutral neck, erect trunk, hips and knees flexed to 90 degrees, and feet in contact with the floor. Patients were allowed to grip a safety bar, or assistants held their trunk, if necessary. Patients were assessed in lateral and/or anterior-posterior view using a fluoroscope (Shimavision 3500 HG, Shimadzu, South Korea). MBSS was performed according to the modified Logemann protocol; nectar-thick liquid was first evaluated and pureed, yielding semi-solid and solid test diets, and finally juice-thick liquid. WSS was performed using Hwang’s protocol, consisting of juice-thick liquid and thenpureed, semi-solid and solid diets. The amount of each material depended on the results of preceding analyses. 

SECTION: Materials
1.
TITLE: PCR for amplification of bark beetles or fungal DNA
AUTHORS: Demian F Gomez, You Li, Caroline Storer

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The purpose of this protocol is to conduct PCR of bark beetles or fungal DNA.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]