eamag/protocols-with-steps huggingface dataset:
TITLE: Protocol for Electronic von Frey
AUTHORS: Lani Tieu, Lauren Smith, Olivier George

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The von Frey method measures mechanical sensitivity and allodynia. This protocol outlines the procedure for assessing the mechanical sensitivity threshold in rats using the electronic von Frey.  </div></div>

[STEPS]
?. [Von Frey Setup]
Remove metal grid and clear boxes off the elevated platform
?. [Von Frey Setup]
Place touch stimulator on testing surface and remove the needle cap
?. [Von Frey Setup]
Place the metal grid back on the legs making sure the grid does not touch the needle
?. [Von Frey Setup]
Place the clear boxes back on top of the grid making sure the opaque sides are placed to prevent animals from seeing each other
?. [Von Frey Setup]
Switch on the Ugo Basile® Dynamic Plantar Aesthesiometer 37450 machine
?. [Von Frey Setup]
Press F4 ("ESC") once, then F1 twice ("OPR" then "TST" ) to display the testing screen
?. [Testing ]
Remove the lid and carefully place one rat per clear box, then place the lids on
Up to 6 animals can be tested at onceIf the animals are weighing down the grid causing it to touch the needle, take out 1 or 2 animals and test less animals at a time and spread them evenly across the gridThe touch stimulator should move across the surface freely without the needle touching the grid while testing a set of animals
?. [Testing ]
Allow rats to habituate in the clear boxes for 10 minutes
?. [Testing ]
Measure the left hind paw first for each animal, then go back and measure the right hind paw for all animals.
?. [Testing ]
Position the needle under the center of the hind paw, then press the button on the touch stimulator to begin the force measurement
Make sure the needle goes through the net and actually touches the paw.
?. [Testing ]
When paw withdrawal occurs due to pain, the needle will retract and the force and time will display on the machine. Record these measurements.
The animal must be still during the application of the needle tip.
?. [Testing ]
Measure each hind paw for all animals for Trial 1. Then repeat for Trial 2, and Trial 3. Once you have 3 Trials you are done.
If an animal is walking or moving not because of pain, move on to the next animal and come back to it later. Wait at least 2 minutes before retesting the same paw.
?. [Testing ]
After testing is complete, return animals to home cages
?. [Testing ]
Clean the clear boxes and grid before testing another set of animals
?. [Cleanup]
Clean all parts of the electronic von Frey (boxes, grid, needle) and make sure to recap the delicate von Frey needle.
TITLE: 5 uM DAPI cell staining solution
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for preparation of 5 µM DAPI cell staining solution that is applicable for staining adult mouse brain sections.</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: Cell Viability Assessment
AUTHORS: Bo Am Seo, Valina L. Dawson, Ted M. Dawson, Hanseok Ko

[DESCRIPTION]
Assess neuronal viability.

[STEPS]
1.     
Stain neurons with Hoechst 33342 (7 micromolar (µM)) and propidium iodide (PI) (2 micromolar (µM)) (Invitrogen) at 37 °C for 5 min.

2. Capture images by a microscope and dead cells are automatically counted by Axiovision 4.6 software (Carl Zeiss).
3. Subject background signals of the control group intensity.
4. Calculate the neuronal death percentage by dividing the PI signal by the Hoechst 33342 signal.
TITLE: Expression of MicroRNA-34c in different grades of Oral squamous cell  carcinoma versus normal oral mucosa  (Ex-Vivo Study)
AUTHORS: Marwa   Refaat Mahmoud, Shaymaa Omar Zayed, Shaimaa Ali Hamouda

[DESCRIPTION]
The relevance of miRNAs in cancer was related to the changes in expression patterns and recurrent amplification as well as deletion of miRNA genes in cancers. miR-34c is an important tumor suppressor gene seen to be downregulated in various cancer types, such as neuroblastoma breast cancer, lung cancer and in laryngeal SCC. Previous studies investigating the expression of miR-34c in plasma and tumor tissue in patients, revealed that the high expression of miR-34c was associated with prolonged overall survival and disease-free survival compared with low expression, and it showed the possibility of regarding miR-34c as potential prognostic markers in several cancers.  But to date little is known about the dysregulation of miR-34c in different grades of oral squamous cell carcinoma .The aim of our study is  to evaluate the change of expression level of MicroRNA- 34c in different grades of OSCC versus normal oral mucosal tissues.


[STEPS]
TITLE: Seawater sampling for environmental DNA metabarcoding - rocky intertidal habitats
AUTHORS: Mary McElroy

[DESCRIPTION]
This protocol describes the collection of 1-L seawater samples from rocky intertidal habitats for environmental DNA (eDNA) metabarcoding. This protocol was optimized for sampling long-term rocky intertidal monitoring sites in southern and central California (USA) during daytime low tides for metabarcoding analyses targeting marine macrophytes and macroinvertebrates.

[BEFORE_START]
You must properly decontaminate sampling gear and materials that have been used previously at other sampling locations or were otherwise acquired as non-sterile from a manufacturer. Proper decontamination of field supplies consists of 30-minute (up to overnight) exposure to a household bleach solution diluted to 10% strength in clean water. After bleach application, rinse supplies thoroughly with laboratory grade water when available - clean tap water from a sink, spigot, or garden hose can also be used for rinsing bulky items like boots, waders, etc. Store decontaminated field gear and materials in a clean storage location until you reach the sampling location.

Be aware of the tide cycle and water level at your field site prior to access on the day of sampling.

[STEPS]
1. Wearing gloves, attach a 1L bottle to the sampling pole.
2. Remove the cap and submerge the bottle in seawater at your sampling location. Be careful not to stir up sand or sediment in shallow areas. Fill the bottle completely and rinse once with seawater. Discard this rinse on the shore or sufficiently away from the point of collection. When possible, collect samples from a distance using the telescopic pole to avoid contaminating point of sample collection with trace DNA on footwear.
3. Refill bottle and securely replace the cap. Avoid touching the interior surfaces of the bottle and cap.
4. Detach bottle from the sampling pole and place it in a storage bag, bin, or cooler. Add ice to cooler if storing samples for more than 1 hour before filtration. Keep 1L field blank in the storage bin alongside biological samples.
5. Repeat for 10x 1L bottle samples, collecting samples spread across discrete tide pools and in the surf zone on lower edge of exposed rocky shore. Label samples accordingly.
6. Record the time, water level, and note any relevant site conditions.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Zebrafish, and other small teleosts, are used as experimental models to evaluate human pathologies, including those linked to oxidative and nitrosative stress, inflammation, and nitric oxide signaling. The protocol presents an optimized technique to quantify nitrite levels, in zebrafish tissues, focusing on the brain and head kidney. The protocol is based on the classical Griess diazotization reaction (Griess, 1858) method.</p>

[BEFORE_START]
<p>Every biochemical protocol needs to be validated in the laboratory when first introduced. The present protocol describes validation steps that were taken in LaNeC.</p>

[GUIDELINES]
<p>This protocol is intended to quantify nitrite levels in adult zebrafish brain and head kidney tissue samples. It can be adapted for other fish species or other tissues without much hassle; however, tissue amounts may need to be adjusted.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Adapted from http://merenlab.org/2016/06/22/anvio-tutorial-v2/</p>

[BEFORE_START]
<p>The goal of this tutorial is to provide a brief overview of the anvi’o workflow for the analysis of assembly-based shotgun metagenomic data. Throughout this tutorial you will primarily learn about the following topics:</p>
<ul>
<li>
<p>Process your contigs</p>
</li>
</ul>

[STEPS]
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TITLE: Extra Burn Keto challenge For Weight loss
AUTHORS: Keto   Burn DX

[DESCRIPTION]
Extra Burn Keto challenge For Weight loss

[STEPS]
SECTION: Extra Burn Keto challenge For Weight loss
1. Extra Burn Keto is a breakthrough product that has been dubbed the finest weight loss alternative of the year. It contains natural mixes that are helpful to the body and assist the user in achieving improved fitness in a matter of weeks. Several users have provided evidence of the product’s excellent results and efficiency. It inhibits fat deposition in the body and quickly burns all excess fat. Aside from the fat-burning method, there are numerous health benefits.

It penetrates deep into the body and eliminates any undesirable fats deposited there for a long period. It energizes the body while also aiding in weight loss. One gains stamina and strength to work out and get a lean body. Because of its natural components, it has no negative effects on the user’s body. This program contains no harsh chemicals or additions. one can learn more about the product in the following paragraphs, so keep reading.

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It also contains Garcinia Cambogia and raspberry ketones, which aid in fat-burning. All of the ingredients have received clinical approval and have been tested. It is beneficial for body transformation while also promoting good health.Extra Burn Keto
https://www.jpost.com/promocontent/extra-burn-keto-reviews-is-it-legitimate-or-scam-701115
https://www.rcm8.net/community/xenforum/topic/73157/how-does-best-effects-extra-burn-keto
https://lexcliq.com/how-do-extra-burn-keto-work/
https://techplanet.today/post/what-is-extra-burn-keto
https://www.scoop.it/topic/extra-burn-keto-by-micset-talk
TITLE: Nerve Conduction Velocity Tests
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies are performed. The animals are anesthetized with 30/2.5 mg/kg ketamine/xylazine to prevent discomfort. Body temperature is monitered with a dermal temperature probe and maintained at 32º C with a warming lamp during NCV. Body temperature is maintained at 37ºC after NCV using a warming pad to ease animal stress from anesthetic. The nerve studies last less than 30 min per rat or mouse. The electrodes are cleaned with 70% alcohol between animals to maintain pathogen-free status.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bik94kcn.jpg" /></div></div>

[STEPS]
?. [Settings:]
♦ Motor tests   o Duration .02 ms    o Range 25 mA    o low frequency filter 1 Hz    o High frequency filter 10 kHz    o Sensitivity 1 mV    o Time 2 ms/div♦ Sensory test   o Duration .02 ms    o Range 25 mA    o low frequency filter 1 Hz    o High frequency filter 10 kHz    o Sensitivity 50 μV    o Time 2 ms/div
?. [Procedure:]
To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies are performed. The animals will be anesthetized with 30-100/2.5-10 mg/kg ketamine/xylazine to prevent discomfort. Body temperature is monitored with a dermal temperature probe and maintained at 37º C with a warming lamp during NCV. Skin temperature is maintained at 34º C during NCV using an infrared thermometer. Body temperature is maintained at 37ºC after NCV using a warming pad to ease animal stress from anesthetic. The nerve studies will last less than 30 min per rat or mouse. The electrodes are cleaned with 70% alcohol between animals to maintain pathogen-free status.Sciatic-tibial motor NCV is determined by stimulating distally at the sciatic notch and distally at the ankle via bipolar electrodes with supramaximal stimulation. The conduction velocity is calculated using two the points of stimulation along the nerve and measuring the resultant onset latency and distance. Sensory NCV is determined by stimulating the sural nerve distally at the ankle via bipolar electrodes with supramaximal stimulation and recording at the fourth and fifth digit. The conduction velocity is calculated using the onset latency and distance.Tail motor latency is determined by stimulating distally along the tail at a recorded distance of 3 cm. The onset latency is used for the latency measurement. Tail sensory NCV is determined by stimulating proximally along the tail at a recorded distance of 3 cm. The conduction velocity is calculated using the onset latency and distance.
TITLE: QUINT Workflow Appendix
AUTHORS: Michael X. Henderson

[DESCRIPTION]
This protocol contains additional details for the modified QUINT workflow.

[STEPS]
SECTION: QuPath: Setting Stain Vectors
1. Draw a small rectangle over a characteristic DAB or hematoxylin stain.
SECTION: QuPath: Setting Stain Vectors
2. On Image tab, double-click the Stain you would like to set. Click Yes on the prompt.
SECTION: QuPath: Setting Stain Vectors
3. Give the new settings a unique name (Project ID). Click OK. 
 

SECTION: QuPath: Setting Stain Vectors
4. This has only saved the parameters for this particular image. To change all images in a project, go to the Workflow tab. This script can also be added to an analysis script and run at the same time.
SECTION: QuPath: Setting Stain Vectors
5. “Set color deconvolution stains” should be the last Command. Click on Command, verify that the parameters are correct, and click “Create script.”
SECTION: QuPath: Setting Stain Vectors
6. In the Script Editor, click Run > Run for Project.
SECTION: QuPath: Setting Stain Vectors
7. Move all images from Available to Selected, and click “OK.” Modified setting from Aperio Scanner:

Hematoxylin: 0.667, 0.658, 0.349
DAB:0.324, 0.547, 0.772
DAB:0.485, 0.616, 0.620
SECTION: QuPath: Pixel Classifier Generation
8. To create a new pixel classifier for segmentation:

It is easiest if the output segmentation from QuPath is black. Click on the “Annotations” tab, 
double click on “Positive” and change the color to black. Click “OK”.
SECTION: QuPath: Pixel Classifier Generation
9.  Use the rectangle tool to box an are you can use to test the classifier.
SECTION: QuPath: Pixel Classifier Generation
10. Classify > Pixel classification > Create thresholder
SECTION: QuPath: Pixel Classifier Generation
11. Example settings are below, but can be optimized by project. Generally higher resolution is better, but slower to run and will create larger files. We generally use “Very high (1.01 um/px)” resolution. Give the classifier a name and click “Save”. The classifier will be in your project folder under classifiers and can be moved to another qupath project folder to share with another set of images for consistency.
  
SECTION: QuPath: Pixel Classifier Generation
12. Examples of Staining Artifacts:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Bioinformatic approach to identifying over-represented motifs in the region framing the start codon (25 bp up and downstream) for genes annotated in the three sequenced Labyrinthulomycete genomes (<em>Aurantiochytrium limacinum, Schizochytrium aggregatum, and Aplanochytrium kergulense</em>).</p>

[STEPS]
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TITLE: Severity Grading Table (Appendix B of Phase 3 study of Vaccine Candidate for COVID-19)
AUTHORS: Chris Ockenhouse, Chris Gast, Renee Holt, Jorge Flores

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is Appendix B of "Phase 3 randomized, double-blinded, placebo-controlled trial to evaluate the safety, immunogenicity, and efficacy of </span><span style = "font-weight:bold;">Vaccine Candidate</span><span> against COVID-19 in adults > 18 years of age"</span></div><div class = "text-block">This generic Phase 3 protocol was developed by the PATH team with support of the Bill and Melinda Gates Foundation. The aim of the collection is to share recommended best practices in designing and implementing a Phase 3 study of a COVID-19 vaccine candidate. As Phase 3 trials of different Vaccine Candidates  proceed around the world, following the same protocols will ensure consistency and comparability of the Phase 3 trial results.</div><div class = "text-block"><span style = "font-weight:bold;">Please note</span><span> that this is an evolving document, to be versioned and updated, based on community feedback and new data.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Várias descobertas são publicadas mensalmente com potencial de aperfeiçoar a técnica de analgesia de parto.</p>
<p>A intenção deste protocolo é registrar sistematicamente as mudanças que adotei.</p>

[STEPS]
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TITLE: DNA extraction protocol for saliva samples obtained from Omigene oral OM device using the Qiagen QiaAMP Powerfecal DNA kit for shotgun metagenomic sequencing
AUTHORS: Victoria Meslier, Aymeric David, christian.morabito, mathieu.almeida

[DESCRIPTION]
This protocol describes the procedure for DNA extraction of saliva samples using the Omnigene OM device and the Qiagen QiaMP Powerfecal DNA PRO kit for shotgun metagenomic sequencing. 


[BEFORE_START]
visit the protocols.io for saliva sampling : https://dx.doi.org/10.17504/protocols.io.n92ldm8qol5b/v1

[GUIDELINES]
NA

[STEPS]
SECTION: Salivary Collection Using OMNIgene ORAL OM 501 Device
1. Visit the protocols.io for saliva sampling : https://dx.doi.org/10.17504/protocols.io.n92ldm8qol5b/v1
SECTION: Sample pre-processing
2. Heat the whole OMNIgene Oral OM 501 tubes into an incubator at 50 °C for 2 h.
SECTION: DNA extraction QIAGEN QIAamp Power Fecal Pro
3. Available at https://www.qiagen.com/
TITLE: Cryosectioning fresh frozen tissues for multimodal imaging
AUTHORS: Presha  Rajbhandari, Taruna Neelakantan, Brent R Stockwell

[DESCRIPTION]
This protocol describes sectioning for fresh frozen tissues for multimodal imaging including Hematoxylin & Eosin staining, DESI imaging mass spectrometry, SIMS imaging mass spectrometry and RNAscope imaging.

[GUIDELINES]
Upon finishing cryosectioning, remove the blade and dispose in sharps container before cleaning the cryostat.
Discard tissue remnants in biohazard bag.

[STEPS]
2. Place the tissue block inside the cryostat at -20°C for 30 mins for equilibration. Also, place the slides and substrates inside the cryostat for cooling.
6. Mount the tissue block on the chuck using water (no OCT) and adjust the angle of the chuck as needed.
5. Trim the tissue block at 30-50µm thickness to obtain a flat surface.
7. Position the anti-roll bar appropriately so that it doesn’t scrape the tissue (if placed too far) or roll off of the block (if placed too near).
1. Clean the cryostat surface, anti-roll blade and forceps with ethanol before sectioning.
8. Gently section the tissue at 8µm thickness using an anti-roll bar and place the consecutive sections on individual substrates. Adjust and flatten with a fine tipped brush if necessary. For H&E, DESI and RNAscope imaging, tissue sections are placed on regular or positively charged microscope slides and on gold coated 2.5cm2 substrate for SIMS-imaging.
9. Thaw mount the sections on the target using heat from the finger under the slide.
10. Store at -80C until use.

3. Set the chamber temperature of the cryostat to -20C and object temperature to -18C to -20C
4. Place the blade in place and lock it.
TITLE: UC Davis - Insulin signaling pathway
AUTHORS: Fawaz G. Haj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
This test is designated to determine defects in the insulin signaling pathway, through evaluation of the activation state of the insulin receptor (IR) and its substrate (IRS1/2), as well as downstream target, mainly Akt and MAP kinases.</div></div>

[STEPS]
?. Fast mice for 16 hours by taking away food the day before (3:00pm)
?. The following day, give the mouse an intraperitoneal injection of insulin (10 U/kg) with a 27 G needle.
?. Use cervical dislocation to euthanize mice.
?. Collect maximum blood from portal vein and isolate plasma according to standard protocols or as desired by the P.I.
?. Quickly collect tissues (Liver, Muscle, Adipose and Pancreas). Each tissue should be divided into three portions, one portion should be snap frozen in liquid nitrogen, one portion should be kept into RNA later solution and the third one should be fixed into the appropriate fixative solution. Please note that the whole procedure of tissue collection should be done within 3 minutes maximum.
?. For western blotting, tissues will be lysed into the appropriate lysis buffer.
?. Overall and site-specific tyrosyl phosphorylation of individual components in insulin signaling such as IR and insulin receptor substrates (IRSs) will be determined by immunoprecipiattaion (IP) then probed with phospho-tyrosyl antibodies according to standard protocols. Tyrosine, Serine and/or threonine phosphorylation of other component of the insulin signaling pathway such as Akt and MAP kinases will be determined also according to the standard Western blotting protocols.
?. Note:
Evaluation of the activation state of other component in the insulin signaling pathway is also possible upon special request. Extra charges may apply.