eamag/protocols-with-steps huggingface dataset:
TITLE: Illustrated Protocol for Processing Radiometric Videos Using the Software FLIR Tools : An Approach Applied to a Novel Methodology for Lizard Thermal Exchange Rates Studies
AUTHORS: Frederico M Barroso

[DESCRIPTION]
The novel methodology developed by Mochales-Riaño & Barroso et al. (in press) for experimentally obtaining thermal exchange rates of small lizards through the use of radiometric videos, underpinned the need to devise a thermal video processing protocol to standardize this data collection. This protocol addresses this need as it represents a subsequently optimized version of the processing pipeline used for the aforementioned proof-of-concept study.
This protocol was used to successfully obtain temperature data from several body parts from thermoregulating lizards, enabling internal body temperature to be inferred (as per Barroso et al., 2016 and 2020), as well as providing insights into thermal exchange rates and the patterns of regional heterothermy and temperature redistribution in these animals.
So far, variants of this video analysis protocol have been successfully deployed in several ongoing research projects, on several species of lizards.

[GUIDELINES]
When processing multiple videos for posterior comparison of data, it is recommended that said processing is done by the same person in order to avoid any operator bias. 

[STEPS]
TITLE: Effect of immunonutrition on serum levels of C-Reactive Protein and lymphocytes in patients with COVID-19: Randomized controlled double-blind clinical trial
AUTHORS: Rodrigo Fernandes Weyll Pimentel, Magno Conceição das Merces

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:justify"><span style = "font-weight:bold;">Background:</span><span> Patients contaminated with COVID-19 undergo changes in leukocyte count, respiratory disorders, and an increase in inflammatory substances. To improve the inflammatory condition, some nutrients can be used, including arginine, omega 3 fatty acids and nucleotides. This study aims to evaluate how oral immunonutrient supplements affects serum C-Reactive Protein (CRP) levels and lymphocyte count in patients with COVID-19. </span></div><style>
							  .justify:after {
							    content: "";
							    display:inline-block;
							    width: 100%;
							  }
							</style></div><div class = "text-block"><span style = "font-weight:bold;">Methods:</span><span> In this double-blind clinical trial we randomized 43 adult patients with COVID-19 to receive a standard high protein normocaloric supplement (control) or an immunonutrient-enriched supplement (experiment) for 7 days. The primary outcome was to evaluate changes in total lymphocyte count and serum level of CRP. The assessment of risk and nutritional status of these patients was also performed.</span></div></div>

[STEPS]
TITLE: 10x Protocols: Visium v1 FFPE Deparaffinization, H&amp;E staining, and Imaging -- University of Minnesota  TMCs (CG000409 Rev B)
AUTHORS: IOx Genomics, Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Protocols from 10x Genomics for Visium Spatial Gene Expression v1 chemistry on FPPE samples without the CytAssist component. 

Protocol ID# (CG) and Revision letter provided here: 
    10x protocol CG000409, Revision B -- Deparaffinization, H&E staining, coverslipping, and imaging

Additional protocols added for guidance:
    10x protocol for imaging guidelines 
    10x protocol for tissue preparation guidelines 

Note: These protocols may not be the current version offered by 10x Genomics but were used to produce the specific datasets connected to them. Please review the 10x Genomics support website for the most recent versions of the protocols prior to starting your experiment. 

[STEPS]
2. Additional Protocols/Resources
  
  
https://www.10xgenomics.com/support/spatial-gene-expression-ffpe

1. 10x protocol CG000409, Rev B (Deparaffinization, H&E, etc)
TITLE: Analysis of BDNF protein expression
AUTHORS: Tamiris Silva, Sandra Kalil Bussadori, Raquel Agnelli Mesquita-Ferrari

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Neuroplasticity regards physiological and/or structural changes in circuits in response to changes in patterns caused by an injury and/or environmental influences, resulting in an increase in angiogenesis and synapses. Although neuroplasticity is more commonly associated with the cerebral cortex, all parts of the nervous system, including the spinal cord, demonstrate plasticity, such as synaptic learning and memory mechanisms, dendritic pruning, collateral sprouting and axonal regeneration.Another factor that influences and regulates neuroplasticity is brain-derived neurotrophic factor (BDNF), the most important functions of which are the regulation of synaptogenesis, neuroprotection and increased dendritic branching. BDNF also exerts an influence on the functional and structural aspects of synaptic transmission. Rehabilitation induces neuroplasticity, which is demonstrated by improved functional performance and an increase in BDNF. Protein expression of BDNF will be quantified from the saliva samples using the ELISA technique. A DELUXE HUMAN commercial kit (BioLegend®) will be used and the analysis will be performed following to the manufacturer's instructions.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The SpinSmart PCR purification and gel extraction technologies utilize a lysis buffer containing chaotropic salts that allow DNA to bind to a silica membrane. Binding buffer PCR 1 is added to a PCR reaction or agarose gel slice; the mixture is subsequently loaded directly onto SpinSmart PCR Columns.  Salts, enzymes, and other soluble components are washed away with ethanolic PCR 2 Wash buffer. Purified DNA is eluted using PCR 3 Elution buffer (5 mM Tris/HCl, pH 8.5). </p>
<p> </p>
<p><span style="color: #000339; font-family: Raleway, sans-serif; font-size: 14px; font-style: normal; font-variant-ligatures: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; orphans: 2; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; widows: 2; word-spacing: 0px; -webkit-text-stroke-width: 0px; background-color: #ffffff; display: inline !important; float: none;">Please follow one of the protocols below, depending on whether you are doing a DNA gel extraction or PCR purification.</span></p>

[STEPS]
TITLE: Antibiotics for algaculture
AUTHORS: Victoria Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Antibiotics recipe adapted from Roscoff Culture Collection protocol.</div></div>

[STEPS]
?. [ABX solution]
To  of , add any 3 of:
10 mL
[Ampicillin]
[Gentamicin]
[Kanamycin]
[Neomycin]
[Streptomycin]
It is best not to use all ABX in every solution, but to use a selection of 2-3 so that you can change the combinations in future solutions in case of bacterial resistance developing.
?. [ABX solution]
Mix the solution well and filter using a 0.2 μM filter into a sterile bottle.Store in the fridge at  up to 1 month.
4 °C
TITLE: Illumina denature and dilute
AUTHORS: Angela R.S.  Kruse, Morad C Malek, Jamie Allen, Melissa Farrow, Jeff Spraggins

[DESCRIPTION]
This protocol describes the preparation of a library for Illumina NGS sequencing.

[STEPS]
SECTION: Standard Normalization Method
1. Use the following steps to denature and dilute libraries that have been normalized using standard library quantification and quality control procedures recommended in the library prep documentation.

Follow the steps most appropriate for your library and the version of Illumina Reagent Kit you are using.
Loading concentration can also vary depending on library type and quantification methods.

  
The denaturation steps described in this guide make sure that the concentration of NaOH is not more than 0.001 (1 mM) in the final solution after diluting with HT1. Higher concentrations of NaOH in the library inhibit library hybridization to the flow cell and decrease cluster density.

SECTION: Prepare Reagents
2. Prepare a Fresh Dilution of NaOH

1: Combine the following volumes in a microcentrifuge tube
Laboratory-grade water (800 μl)
Stock 1.0 N NaOH (200 μl)

The result is 1 ml of 0.2 N NaOH.

2: Invert the tube several times to mix.

NOTE
Use the fresh dilution within 12 hours.

SECTION: Prepare Reagents
3. Prepare HT1

1 Remove HT1 from -25°C to -15°C storage and thaw at room temperature.

2 Store at 2°C to 8°C until you are ready to dilute denatured libraries.
SECTION: Denature a 4 nM Library
4. 1 Combine the following volumes in a microcentrifuge tube.
4 nM library (5 μl)
0.2 N NaOH (5 μl)
SECTION: Denature a 4 nM Library
5. Vortex briefly and then centrifuge at 280 × g for 1 minute.
SECTION: Denature a 4 nM Library
6. Incubate at room temperature for 5 minutes.
SECTION: Denature a 4 nM Library
7. Add 990 μl prechilled HT1 to the tube containing denatured library.
The result is 1 ml of a 20 pM denatured library.

Note that the denatured library concentration may vary by Illumina platform.
TITLE: In vitro dephosphorylation
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
In vitro dephosphorylation

[STEPS]
1. 100 μg of α-syn GST fusion protein on beads were resuspended in alkaline
phosphatase (ALP) buffer supplemented with 250 units of ALP (Thermo Scientific
Cat# EF0651). 





2. The mixes were incubated for 60 min or 180 min at 37 °C with
orbital shaking (Eppendorf ThermoMixer F1.5). 

3. Samples were then analyzed by
NuPAGE and immunoblotted or alternatively used in pull-down experiments with
brain lysates.
TITLE: Cryoprotection of mouse brain tissue
AUTHORS: Lauren C. Faget, Thomas Hnasko

[DESCRIPTION]
Fixed brains are submerged in 30% sucrose to cryoprotect prior to freezing and cutting cryosections.

[BEFORE_START]
Following perfusion and post-fixation in 4% ice-cold PFA (for 2 hrs, overnight, or as appropriate for intended use):

[STEPS]
SECTION: Procedure
1. Make 30% sucrose solution by dissolving 30 g of sucrose (BP220-1, Fisher) in 100 mL of 1X PBS (BP3991, Fisher) pH 7.4 

SECTION: Procedure
2. Fill tube (20 mL glass scintillation vials or 15 mL conical centrifuge tubes are classically used) with ~ 10 mL of sucrose and transfer fixed brain tissue into tube, gently mix by inverting tube several times.

SECTION: Procedure
3. Incubate at 4 °Cuntil brain sinks (typically ~2880 min).

SECTION: Procedure
4. Prepare superchilled isopentane (2-methylbutane, M1246, Spectrum) by pouring ~30 mL into 50 ml bottle or beaker. Carefully position beaker on dry ice in a stable manner. Wait until isopentane temperature reaches -30 °C before proceeding to flash freezing.

SECTION: Procedure
5. Rinse brain with chilled 1X PBS.
SECTION: Procedure
6. Dry brain on paper towel by rolling it and gently pressing against paper towel.
SECTION: Procedure
7. Use long-handled forceps to transfer brain to superchilled isopentane. Let brain freeze for ~15 s until completely white and bubbling stops.

SECTION: Procedure
8. Transfer to storage-tube that has been pre-labeled and superchilled on dry ice.
SECTION: Procedure
9. Transfer to -80 °C freezer for long-term storage.
TITLE: UC Davis - Intraperitoneal Insulin Tolerance Test
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">
An intraperitoneal insulin tolerance test or ipITT is designed to determine the sensitivity of insulin-responsive tissues in the rodent. This is determined by measurement of glucose remaining in the circulation over time after a bolus ip insulin injection.</div></div>

[STEPS]
?. Fast mice for 4 h only by taking away food early in the morning (7:00am).
?. Calibrate the glucose meter according to the manufacturer’s instructions.
?. Deprive mice from water then remove approximately 5μl of blood (one drop) from the tail via a tail tip cut and transfer directly onto a glucose indicator strip.
?. Measure blood glucose immediately in a glucometer.
?. Give the mouse an intraperitoneal injection of insulin (0.5 U/kg) with a 27 G needle.
?. Continue to take blood samples from the initial tail cut before the insulin injection and at 15, 30, 45, 60 and 120 min.Between each of these time points, return the mouse to its cage and monitor it every minute.
?. NOTE:
1-The mouse is given an intraperitoneal injection with a 27G needle of insulin. Before performing the experiment, we will have to determine if the mouse strain is insulin resistant or glucose tolerant, so as to avoid giving the wrong dose of insulin.
2-For insulin resistant mice, a higher dose of insulin could be used as they do not respond as well as insulin sensitive mice to insulin. However, we will make sure that the dose of insulin used will not cause hypoglycemia.
3-At the end of the experiment, wipe tail with 70% alcohol and allow drying. Ensure that blood loss from the tail stopped before placing the animal back to its cage.
TITLE: Neural progenitor expansion
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Using the pre-coated PLO-laminin plate, aspirate laminin from 2-3 wells. Remove supernatant from 15 mL conical tube containing 2 wells of neural rosette clusters. See protocol below.
?. Add appropriate amount of neural induction media (NIM) to achieve a final volume of  per well. Pipet clusters up and down breaking them into 1/4 or 1/5 the size of the rosette cluster.
2 ml
?. Add  of neural rosette cluster suspension to each well and incubate at , 5% CO2 and 95% humidified chamber for .
2 ml
?. Examine adherent cells under microscope, aspirate off medium and replace with of fresh neural induction medium daily for 3-5 days or until they reach ~80% confluent.
2 ml
?. Make  of DMEM/F12 supplemented with 10% FBS by adding  of FBS to  of DMEM/F12 to inhibit trypsin activity (termed “Complete Media”).
12 ml
2 ml
10 ml
?. Remove medium and rinse cells with 1 mL/well of DMEM/F12 .
?. Harvest cells by adding 1 mL/well of 0.05% trypsin and incubate at for .
?. Inhibit trypsin activity by addingof DMEM/F12 supplemented with 10% FBS to each well.
4 ml
?. Collect cells in 15mL conical tube. Centrifuge at 750 rpm for .
To minimize bubbles and increase cell yield, add 1mL Complete Media to 15mL conical tube. After trypsinization, add 3mL DMEM/F12 to collect cells and transfer to 15mL conical tube.
?. Aspirate supernatant and resuspend in  of NIM, pipet at least 3 times 
 to break up large clumps.
12 ml
Small clumps and single cells are acceptable to passage.
?. Passage cells to pre-coated PLO-lamin plates (see above) by adding 2 mLs/well of suspended cells for a total of 6 wells.
?. Feed with of fresh NIM daily until they reach 85-95% confluency.
2 ml
?. IMPORTANT: NPCs are best maintained by passaging at a 1:3 dilution. Passaging cells that are more dilute will result in slow growth. Cells will typically become confluent after 2-4 days when plated as a 1:3 dilution. If NPCs are observed to slow in their growth, passage cells and replate without dilution.
TITLE: Mouse sample collection for metabolomics studies
AUTHORS: Livia Hecke Morais

[DESCRIPTION]
Protocol used in the Mazmanian lab for collecting brain and gut tissues and plasma from mouse for metabolomics

[STEPS]
SECTION: Samples collection for metabolomics studies
1. At 4 months of age, mice were sacrificed by
decapitation
SECTION: Samples collection for metabolomics studies
2. The brain was rapidly removed from the skull and placed in an ice-chilled stainless steel coronal
matrix
SECTION: Samples collection for metabolomics studies
3. Brain tissue was sectioned in slices of approximately 1 mm
SECTION: Samples collection for metabolomics studies
4. Substantia nigra, striatum, motor cortex(referred to as cortex) and caudal brainstem (referred to as brainstem) were dissected within three minutes using a brain atlas as reference1 
SECTION: Samples collection for metabolomics studies
5. Gut tissue and contents were dissected on an ice-chilled stainless steel dissection
tray
SECTION: Samples collection for metabolomics studies
6. All tissue samples were weighed, stored in and stored at Precellys‱ 2 mL Soft Tissue Homogenizing Ceramic Beads tubes (Catalog number: 10011152), snap-frozen in dry ice and stored at -80ºC
until processing
SECTION: Samples collection for metabolomics studies
7. Trunk blood was collected in BD Vacutainer‱ Hemogard‱ Closure Plastic K3-Edta Tubes (Catalog number: 15349700) and kept at room temperature before plasma separation

SECTION: Samples collection for metabolomics studies
8. Plasma was separated by centrifugation at 2,500 x g for 10 minutes 
SECTION: Samples collection for metabolomics studies
9. Plasma was transferred to a pre-cooled collection vial Thermo Scientific Pierce 1.5 mL Capacity Microcentrifuge Tubes (Catalog number: 69715) and stored at -80ºC until processing
SECTION: Samples collection for metabolomics studies
10. References: 
1. Paxinos, G. and Franklin, K.B.J. (2001) The Mouse Brain in Stereotaxic Coordinates. 2nd Edition, Academic Press, San Diego.
TITLE: Yale - Blood Albumin
AUTHORS: Gary Cline, John Stack

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of albumin in blood, plasma, and serum. Albumin is measured as its conjugate with bromocresol green monitored at 600 nm.</div></div>

[STEPS]
?. Calibrate Cobas for Albumin analysis by running an albumin standard, assayed control serum 1 and assayed control serum 2.
?. Sample handling as performed by the Cobas Mira Plus.      a) Pipette 2µL of sample into a cuvette slot.      b) Add 250 µL of Albumin reagent and mix.      c) Mixture is incubated at 37˚C and spun for 10 minutes.      d) Absorbance is measured at 600 nm.
TITLE: No Title
AUTHORS: 

[STEPS]
SECTION: Preparation of LBv2 medium
?. 
SECTION: Preparation of glycerol stock
?. 
SECTION: Preparation of glycerol stock
?. 
SECTION: Preparation of glycerol stock
?. 
SECTION: Preparation of glycerol stock
?. 
SECTION: Preparation of glycerol stock
?.
TITLE: TEV Protease
AUTHORS: New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">TEV Protease is a highly specific cysteine protease that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues.</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">Removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins</li><li style = "counter-reset:ol0;list-style-type:disc;">Optimal activity and stability for up to 24 months</li><li style = "counter-reset:ol0;list-style-type:disc;">Active in a wide range of buffers; optimal activity between pH 6.0 and 9.0.</li><li style = "counter-reset:ol0;list-style-type:disc;">High substrate specificity with no non-specific proteolysis</li></ul></div><div class = "text-block"><span>TEV Protease (</span><a href="https://www.neb.com/products/p8112-tev-protease" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">NEB# P8112</span></a><span>) is recommended for cleavage of a His-tag following purification with NEBExpress</span><span style = "vertical-align:super;">®</span><span> Ni-NTA Magnetic Beads, Ni Spin Columns or Ni Resin. First, the expression vector must be designed to contain a TEV site between the His-tag and the protein. </span></div></div>

[STEPS]
?. [Typical Reaction Conditions for TEV Protease]
Dialyze the protein against , .
[Tris-HCl]
?. [Typical Reaction Conditions for TEV Protease]
Determine the protein concentration.
?. [Typical Reaction Conditions for TEV Protease]
Combine  (if necessary) to make a  total reaction volume.
[of protein and H20]
45 µl
?. [Typical Reaction Conditions for TEV Protease]
Add  to make a  total reaction volume.
[TEV Protease Reaction Buffer (10X)]
50 µl
?. [Typical Reaction Conditions for TEV Protease]
Add .
[TEV Protease]
?. [Typical Reaction Conditions for TEV Protease]
Incubate at  for  or at .
30 °C
4 °C
TITLE: KU Leuven Exp Urology - Urodynamics in Female MInipigs
AUTHORS: Yodi Soebadi, Marko Bakula, Lukman Hakim, Robert Puers, Dirk De Ridder

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span><span style = "font-weight:bold;"> </span></div><div class = "text-block">This protocol provides information for the invasive urodynamic measurement of bladder pressure, by urethral catheter as well as wireless device (bladder pill).</div></div>

[STEPS]
?. Preparation: Bring machine and all consumables to room
?. Fill calibration tube with saline
?. Check calibration with tube
?. Open charge switch AND flush baloon
?. Measurement:  Isolate minipig for study, bring in transport cart & administer sedation: Ketamine 15 mg/kg (Nimatek 100 mg/ml) Xylazine 2 mg/kg (Xyl-M 2%); (at 60-80 kg, 6-8 ml of Nimatek, 1-1.5 ml of xylazine>>
?. Wait 10-15 minutes outside of cage until minipig sedated
?. Place minipig prone at caudal end,.
?. Prepare urethral silicone cath, gel, tape.
?. Insert single and triple channel catheter in urethra, insert rectal catheter
?. Put belt or frame around midsection
?. Set zero on UDS machine then charge catheter.
?. Put tape around charge switch
?. Tape plastic around posterior and tape funnel
?. Wait for animal to wake up (start eating) and start filling at 10% of voided volume (at volume more than 500 ml fill 50 ml/minute)
?. Repeat up to 5 voids
?. At end of procedure, anesthetise and extract pill.