eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The use of genetically encoded ‘self-labeling tags’ with chemical fluorophore ligands enables rapid labeling of specific cells in neural tissue. To improve the chemical tagging of neurons, we synthesized and evaluated new fluorophore ligands based on Cy, Janelia Fluor, Alexa Fluor, and ATTO dyes and tested these with recently improved <em>Drosophila melanogaster</em> transgenes. We found that tissue clearing and mounting in DPX substantially improves signal quality when combined with specific non-cyanine fluorophores. We compared and combined this labeling technique with standard immunohistochemistry in the Drosophila brain.</p>

[BEFORE_START]
<p><strong>Reagents and Supplies</strong></p>
<p><strong> </strong></p>
<ul>
<li>Cy2 Goat α- Jackson Immuno Research. # 115-225-166</li>
<li>Cy3 Goat α- Jackson Immuno Research. # 111-165-144</li>
<li>ATTO 647N Goat α-Rat IgG (H&amp;L) Antibody. Rockland. # 612-156-120</li>
<li>DPX Mountant for Microscopy. Electron Microscopy Sciences. # 13512, 500 mL</li>
<li>Ethanol, ACS reagent, &gt;99.5% (200 proof). Sigma Aldrich. # 459844-1L</li>
<li>GS – Goat Serum. Life Technologies. # 16210-064, 100 mL</li>
<li>Kodak Photo-Flo 200 Solution. Electron Microscopy Sciences. # 74257</li>
<li>Cy2 Snap ligand, Luke Lavis, JRC</li>
<li>PBS - Phosphate Buffered Saline, 1X<strong>. </strong> # 21-040</li>
<li>PFA – Paraformaldehyde. 20% PFA. Electron Microscopy Sciences. # 15713-S</li>
<li>Poly-L-Lysine. Sigma Aldrich. # P1524-25MG</li>
<li>Protein LoBind Microcentrifuge Tubes. Eppendorf. # 022431102</li>
<li>Rat α-FLAG Tag (DYKDDDDK Epitope Tag). Novus Biologicals. # NBP1-06712</li>
<li>Rabbit α-HA Tag. Cell Signal Technologies. # 3724S</li>
<li>S2 – Schneider’s Insect Medium. Sigma Aldrich. # S01416</li>
<li>Triton X-100. Sigma Aldrich. # X100</li>
<li>Fisher Scientific. # X5-500</li>
</ul>

[GUIDELINES]
<ul>
<li>All tissues and solutions are at room temperature (RT), unless noted. Always protect tissue from light exposure.</li>
<li>For details on dissection and fixation see FlyLight Protocol - Adult Dissection and 2% Fixation.</li>
<li>For mounting and embedding instructions refer to FlyLight Protocol – DPX Mounting.</li>
<li>Protocols at <a href="https://www.janelia.org/project-team/flylight/protocols" target="_blank" rel="noopener noreferrer">https://www.janelia.org/project-team/flylight/protocols</a></li>
</ul>
<ul>
<li>Reporter Genotype: w; brp-SNAP / CyO; pJFRC225-5xUAS-IVS-myr::smGFP-FLAG in VK00005, pJFRC51-3xUAS-Syt::smGFP-HA in su(Hw)attP1</li>
<li>For details on polarity constructs, please refer to Aso et al. 2014. <a href="http://elifesciences.org/content/3/e04577" target="_blank" rel="noopener noreferrer">http://elifesciences.org/content/3/e04577</a></li>
<li>For details on brp-SNAP chemical tagging, please refer to Kohl, et al., 2014. <a href="http://www.pnas.org/content/111/36/E3805.long" target="_blank" rel="noopener noreferrer">http://www.pnas.org/content/111/36/E3805.long</a> doi: 10.1073/pnas.1411087111</li>
</ul>

[STEPS]
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TITLE: Analysis of immofluorescence images in ImageJ
AUTHORS: rachel.bates

[DESCRIPTION]
Analysis at a single cell level of images taken on confocal

[STEPS]
1. Import image into ImageJ as a TIFF file. 
2. Set scale for Image by going to Analyze --> set scale. Set distance in pixels and unit of length to appropriate values for objective images were collected with. 
3. Convert image to 8 bit going to Image --> type --> 8 bit. 
4. Open up ROI manager using Analyser --> tools --> ROI manager. 
5. Use freehand selection tool to carefully draw around the outline of each cell. For each cell outline add to ROI manager by selecting ADD. 
6. Set up measurement parameters in Analyse --> set measurements --> select AREA, MIN & MAC GREY AREA, INTEGRATED DENSITY, MEAN GREY VALUE AND LIMIT TO THRESHOLD. 
7. If a multichannel image convert to composite. 
8. Go to IMAGE --> ADJUST --> THRESHOLD. From drop down menu select intermodes. 
9. Use a consistent threshold for all images,set to threshold and in ROI manager select MEASURE. 
10. Calculate a background fluorescence of each image aquired by drawing 3 ROI not including a cell. Take a measurement of background using the same threshold settings. 
11. Calculate corrected total cell fluorescence using = integrated density measurement - (cell area * mean average background fluorescence).
TITLE: Purification of lysosomes using dextran-conjugated SPIONs
AUTHORS: William  Hancock-Cerutti, Arun  Kumar Tharkeshwar, Shawn  M Ferguson, Pietro  De Camilli

[DESCRIPTION]
This method describes the usage of dextran-conjugated superparamagnetic iron nanoparticles (SPIONs) to purify a lysosome fraction from cultured cells.

[STEPS]
TITLE: Copy of Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-Salmonella antibody in layer hen&#39;s egg yolks.
AUTHORS: Angel Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Enzyme-linked immunosorbent assay (ELISA) for studying the presence of anti-</span><span style = "font-weight:bold;font-style:italic;">Salmonella</span><span style = "font-weight:bold;"> antibody in layer hens was a reproducible and feseable test used to meassure IgY development after vaccination. </span></div></div>

[STEPS]
?. U-shaped bottom's ninety-six well polystyrene microplate purchased at Sigma-Aldrich, St. Louis USA was incubated with (2 µg/well) of the LPS (Sigma –Aldrich) from Salmonella Typhimurium in coating buffer (overnight at 4 ºC.)
?. The microtiter plates was washed four times, with 10 % PBS-Tween-20.
?. The microplate was blocked with 3% non-fat milk in PBS (25 µl/well).
?. The microplate was incubated 1 hr at RT .
?. The microplate was washed four times.
?. Then a 50 µl aliquot of the egg yolk (Ig)Y solutions in a concentration of 1.25 mg/ml was added in triplicate. The IgY concentration was assessed by ELISA and sample were titrated with sample buffer until it got the expected IgY concentration.
?. After incubating for one hour at RT, the microplate was washed four times.
?. Fifty (50 µl) of the anti-IgY-HRP conjugate (Sigma-Aldrich) diluted to 1:30000 with conjugate diluent was  added into each well.
?. The microplate was incubated for 1 hr at RT.
?. Then, the microplate was washed four times.
?. Fifty (50 µl) of tetramethylbenzidine (TMB, Sigma-Aldrich) was added into each well.
?. The microplate was further incubated for 15 minutes in the dark.
?. Fifty (50 µl) 3M HCl was added to the microplate for stopping  the reaction.
?. After that, reaction  color development was measured with a microplate reader (Synergy™ Neo Hybrid Multi-Mode Microplate Reader).
?. The cut-off point was an OD of 0.51, and it was calculated from the XOD of the negative x 2 SD. This ELISA tested triplicates of a total of 90 IgY preparations.
TITLE: 4AA Mix
AUTHORS: Erçağ Pinçe

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">4 amino acid mixture for MMH1 </div></div>

[STEPS]
?. [Mix the ingredients ]
40 mL ddH2O(milliQ)620 mg L-Histidine [100mM]596 mg L-Methionine [100mM] 524 mg L-Leucine [100mM]476 mg L-Threonine [100mM]Stir for overnight at 37.5C. Adjust pH to 7.0.Filter sterilize and store in the cold room(+4°C)
TITLE: Evaluation of elasticity of the pancreas by Acoustic Radiation Force Impulse (ARFI) in acute pancreatitis patients: A systematic review and meta-analysis (protocol)
AUTHORS: Kazuma Rifu, Jun Watanabe, Hideki Sasanuma, Nobuyuki Taniguchi

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">Acute pancreatitis (AP) is characterized by inflammation of the pancreas(1). The morbidity of AP is reported about 5-80/100000 people/year (2). An increase in the incidence of AP has been also reported (3-9). The mortality rate has been reported to be 1.7-8.4% (10, 11), and AP still remains a life-threatening disease. Clinical symptoms and elevated levels of pancreatic enzymes constitute the main diagnostic features of this condition.(12, 13) Early diagnosis and treatment are important, and reliable imaging techniques are required for a diagnosis of acute pancreatitis.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Acoustic radiation force impulse (ARFI) elastography is a promising ultrasound-based imaging modality for the evaluation of tissue stiffness including in the diagnosis of AP (14). Studies using the ARFI elastography evaluated different abdominal organs(15), especially in pancreatic disease, such as AP, chronic pancreatitis, or pancreatic carcinoma, relevant higher shear wave velocities than in healthy pancreas parenchyma are described(1, 16, 17). In this study, we evaluated acoustic radiation force impulse (ARFI) elastography, Virtual Touch tissue quantification (VTQ; Siemens Medical Solutions, Mountain View, CA),to diagnose acute pancreatitis. The consistency of the pancreatic parenchyma usually becomes stiffer in acute pancreatitis as compared to the healthy pancreas, which is identifiable with Share Wave Elastography (SWE), including ARFI (18, 19). However, studies using elastographic techniques in patients with acute pancreatitis are conflicting(19).One prospective study failed to find significant differences in pancreatic shear wave velocities between patients with AP and healthy volunteers(14, 19). Three other studies showed significantly higher pancreatic shear wave velocities in patients with acute pancreatitis compared to persons with the normal pancreas(1, 18-20).</div></div><div class = "text-block">The present study will aim to assess the effectiveness of ARFI elastography in the form of ARFI-VTQ imaging in characterizing and differentiating normal pancreas and acute pancreatitis.</div></div>

[STEPS]
TITLE: Metabarcoding PCR Protocol
AUTHORS: Mirayana Marcelino Barros, Tiago Pereira, Alejandro De Santiago Perez, Hunter Powell, Jenna Brown

[DESCRIPTION]
18S Metabarcoding PCR Protocol using Invitrogen Platinum Hot Start PCR 2X Master Mix (REF 13000014) buffer.

[STEPS]
SECTION: Preparing the PCR tubes
1. Label PCR tubes and place them under a UV light for 10 min.

SECTION: Metabarcoding PCR
4. Transfer 18 µL of the master mix to each PCR tube.

SECTION: Metabarcoding PCR
5. Transfer 5 µL of the DNA template to each PCR tube.

SECTION: Metabarcoding PCR
6. Transfer 2 µL of the Metabarcoding Primer to the assigned PCR tube. Immediately place primers on a PCR Cooler Rack after use. 

SECTION: Metabarcoding PCR
7. Mix the samples well using a vortex, then briefly centrifuge them using a microcentrifuge or platefuge to ensure all contents are at the bottom.
SECTION: Metabarcoding PCR
8. Place samples in a thermo cycler and set to a PCR protocol. Set the thermo cycler to use a heated lid and wait until the lid and block are close to the temperature. See 8.1 for thermo cycler specifications.
Tip: In the Bik Lab, select protocol “METAB-HS” on thermo cycler.
SECTION: Metabarcoding PCR
9. Close the thermo cycler lid well.
SECTION: Preparing the master mix
2. Add 10 µL of Invitrogen Platinum Hot Start PCR 2X Master Mix (REF 13000014) buffer and 8 µLof molecular water to a 1.5 mL tube PER NUMBER OF SAMPLES i.e., for 4 samples, add 40 uL of buffer and 32 uL of molecular water to a 1.5 mL tube.
SECTION: Preparing the master mix
3. Mix well using a vortex, then centrifuge.
SECTION: Metabarcoding PCR
8.1. METAB-HS Protocol: 
Lid: 105°C 
Volume: 25 μL
1. 94°C, 3:00
2. 94°C, 0:45
3. 50°C, 1:00
4. 72°C, 1:30
5. GOTO step 2, 34X
6. 72°C, 10:00
7. 4°C, ∞
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Bloodstream infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are associated with treatment failure and increased mortality. Detection of CPE from blood cultures (BC) by standard methods takes 16-72 hours, which can delay the initiation of appropriate antimicrobial therapy and compromise patient outcome. We developed and evaluated a new method for the rapid detection of carbapenemases directly from positive BC using a new multiplex immunochromatographic test (ICT), the RESIST‑3 O.K.N. ICT® (Coris BioConcept, Gembloux, Belgium). It was originally developed for the detection of OXA-48-like, KPC and NDM carbapenemases from bacteria grown on solid media. <br /><br /></p>

[BEFORE_START]
<p>Prepare</p>
<p>10% SDS (can be stored for several weeks, but make sure that it has not crystallized)</p>
<p>0.5 M ZnSO<sub>4 </sub>(can be stored for months)</p>
<p> </p>
<p>Other reagents required:</p>
<p>double distilled water ddH<sub>2</sub>0</p>
<p>RESIST-3 O.K.N. ICT (Coris BioConcept, Gembloux, Belgium)</p>
<p>1.5 ml reaction tubes (e.g. Eppendorf)</p>
<p> </p>

[GUIDELINES]
<p>Follow guidelines for good laboratory practice (GLP)</p>

[STEPS]
?. 
SECTION: Hemolysis of red blood cells
?. 
SECTION: Washing of bacteria
?. 
SECTION: Lysis of bacteria
?. 
SECTION: Loading of the immunochromatographic assay
?. 
SECTION: Reading of the immunochromatographic assay
?. 
SECTION: Optional step - incubation with ZnSO4 for improved performance with NDM-producing Enterobacteriaceae (advanced protocol)
?. 
SECTION: Tipps & Troubleshooting
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SECTION: Results
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TITLE: Water preservation for flow cytometry
AUTHORS: Melissa B Duhaime, Rachel Cable

[DESCRIPTION]
This method of preservation for flow cytometry was adapted from the TARA Oceans protocol for bacterial and viral flow cytometry.

This version of the protocol was edited by Rachel Cable for use in EXPORTS 2018 cruises and for field work in Microbes in the Wild: Advanced Environmental Microbiology Lab (EEB 447) course at University of Michigan Biological Station and on campus. 



[STEPS]
SECTION: Glutaraldehyde preparation
1. For 1.5 mL sample volume:
Aliquot 7.5 µL of 25% aqueous gluteraldehyde into each cryovial. Store aliquoted glutaraldehyde at 4°C until use.
 
 
 
SECTION: Sample preservation
4. Using a sterile 10-ml syringe (see bleaching protocol in Step 2 if syringe is being reused) and the prepared filter cartridge, aliquot 1.5 mL of your 20 um-filtered seawater sample into each cryovial. Invert to mix. Label your cryovials with a ultra fine point Sharpie marker.
SECTION: Sample preservation
5. Incubate at room temperature in the dark for 15 minutes.
15 min
SECTION: Sample preservation
6. Snap-freeze the samples in liquid nitrogen. Store at -80°C.
SECTION: Filtration preparation
2. Clean Swinnex filter holders by soaking in 10% bleach for 1-24 hrs. Then rinse thoroughly with MilliQ water. Allow to dry completely or use immediately. Syringes can also be bleached in the same manner and reused to reduce plastic waste.
 

 
SECTION: Filtration preparation
3. Using sterilized forceps, place a 20 um nylon net filter in the filter holder and tightly screw the filter holder together.
TITLE: Estimating the cost of HIV/AIDS and TB services at health facilities in Cambodia
AUTHORS: kouland thin, Benjamin Johns

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In Cambodia, HIV/AIDS and TB have historically been financed by donors, the Ministry of Health, and</div><div class = "text-block">households (via out-of-pocket spending). Donor funding for HIV/AIDS and TB is declining, the</div><div class = "text-block">government is struggling to generate new resources dedicated to the prevention or treatment of these</div><div class = "text-block">diseases. However, in recent years Health Equity Funds (HEFs) have begun to provide financing for</div><div class = "text-block">HIV/AIDS and TB services. Upcoming social health protection reforms increase the likelihood that new</div><div class = "text-block">social health insurers, e.g. the National Social Security Fund (NSSF), will also cover HIV/AIDS and TB</div><div class = "text-block">services, as has been done in other countries in the region, including Vietnam, Thailand, and the</div><div class = "text-block">Philippines. The NSSF currently does not clearly specify that HIV/AIDS and TB services are included in the</div><div class = "text-block">benefit package; rather the NSSF health benefit package specifies that it covers ‘treatment and care</div><div class = "text-block">services with medical professional techniques’.</div><div class = "text-block">The purpose of this study will be to understand the unit costs of delivering HIV/AIDS and TB services,</div><div class = "text-block">identify the major components of costs, sources of funding, and to inform HEF (and eventually NSSF)</div><div class = "text-block">reimbursement rates. In the short term, the results will be used to build an actuarial cost and financing</div><div class = "text-block">model projecting resource needs and likely sources of funding for HIV/AIDS and TB services over 5 years.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[GUIDELINES]
SOB medium<br />
<ul>
<li>1.0% Bacto-tryptone</li>
<li>0.5% Bacto-yeast extract</li>
<li>10.0 mM NaCl</li>
<li>2.5 mM KCl</li>
<li>10.0 mM MgCl<sub>2</sub></li>
<li>10.0 mM MgSO<sub>4</sub></li>
</ul>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>    A method for concentrating aquatic microbes for single-cell analysis by Raman microspectroscopy. Modified from Hewes and Holm-Hansen (1983): A Method for Recovering Nanoplankton from Filters for Identification with the Microscope: The Filter-Transfer-Freeze (FTF) Technique. Limnology and Oceanography. 28(2): 389-394.</p>
<p> </p>
<p>     Many materials commonly used in microscopy to examine individual microorganisms contribute contaminating Raman scattered emissions, e.g., most types of membranes and filters, glass slides, cover slips, immersion oil, glycerol, etc. and are therefore unsuitable for this application. We developed a protocol that enables single-cell analysis while minimizing extraneous Raman scattered photons.  The technique transfers cells from popular polycarbonate membranes to mirror-finished 304 stainless steel slides (1x3x0.0235') supplied by Stainless Supply® (Monroe, NC USA).</p>
<p> </p>

[BEFORE_START]
<p>Additionally, a frozen aluminum block (previously stored at -80°C) is necessary as well as typical lab supplies such as pipettors and forceps.</p>
<p> </p>
<p><strong>Note</strong>: Polycarbonate membrane filters of various pore sizes can be used. </p>

[STEPS]
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TITLE: Anti-Neu5Gc Antibody Kit Protocol - ELISA
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is essentially the same as a standard ELISA protocol, with one major exception - all animal compounds must be avoided in buffers, etc. because they contain Neu5Gc, which will interfere with antibody activity and result in misleading data. Essentially, the glycoconjugate to be evaluated is immobilized on an ELISA plate and exposed to a primary antibody. The antibody provided in this kit has been shown to identify as little as 5pmol of Neu5Gc per microgram glycoprotein, which is at or below the current detection limit for conventional DMB HPLC analysis. An advantage of the ELISA procedure is that it confirms the presence of Neu5Gc on the glycoconjugate of interest, but use caution, as it also reacts with solution/media that contains glycoconjugate.</div></div>

[STEPS]
?. Coat the 96-well ELISA plate with the target molecule in 50mM sodium carbonate-bicarbonate buffer, pH 9.5 (up to 1µg/well in 100µl carbonate buffer, in triplicate) for at least 2 hours at ambient temperature or overnight at 4°C.
?. Wash the wells 3 to 5 times with PBS.
?. Block wells with PBS-T (200µl/well) for 1 hour at room temperature.
?. Add the anti-Neu5Gc antibody (or the negative control antibody), diluted in PBS-T, to the appropriate well(s) and incubate for 2 hours at room temperature. Note: The resuspended primary and control antibody dilution range is between 1:1,000 and 1:10,000. The optimal dilution will need to be determined by the researcher. The suggested dilution for the secondary antibody is 1:5,000, but this should also be optimized for the particular application.
?. Wash wells as before with PBS-T.
?. Add enzyme-conjugated secondary antibody (donkey-anti-chicken antibody, 1:10,000 in PBS) to each well and incubate for 1 hour at room temperature.
?. Wash wells again with PBS-T.
?. Add enzyme substrate, allow the wells to develop; quench the reaction if necessary.
?. Read absorbance on an ELISA plate reader at the appropriate wavelength.
TITLE: Effect of Multiple Biofortified Food Crops-based Meals on Micronutrient Status and Immune Function in The First 1000 Days of Life in India: Study Protocol for a Randomized Controlled Feeding Trial v2.0
AUTHORS: Saurabh Mehta, Bryan Gannon, Camille E. Jones, Laura S. Hackl, Shoba A. Udipi, Varsha Thakker, Aparna Thorat, Wesley Bonam, Jere D. Haas, Julia Finkelstein

[STEPS]
TITLE: Rachael Ray CBD Gummies - Rachael Ray CBD Gummies , Quit Smoking and 100% Pain Relief!
AUTHORS: RachaelRaycost  

[DESCRIPTION]
The extraction of this is finished by dependable sources that continually utilize moderate extraction measures. Your body benefits from CBD. It urges you to quiet down and get the recuperation hit. It carries general wellbeing and thriving to you as a client.

[STEPS]
TITLE: MojoSort™ Human CD56 Nanobeads Column Protocol
AUTHORS: Ken Lau

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">MojoSort™ Human CD56 Nanobeads Column Protocol</span><span></span></div></div>

[STEPS]
?. [Protocol]
Prepare cells from your tissue of interest or blood without lysing erythrocytes.
?. [Protocol]
In the final wash of your sample preparation, resuspend the cells in MojoSort™ Buffer by adding up to 4 mL in a 5 mL (12 x 75 mm) polypropylene tube.Note: Keep MojoSort™ Buffer on ice throughout the procedure.
?. [Protocol]
Filter the cells with a 70 µm cell strainer, centrifuge at 300xg for 5 minutes, and resuspend in a small volume of MojoSort™ Buffer. Count and adjust the cell concentration to 1 x 108cells/mL.
?. [Protocol]
Aliquot 100 µL (107cells) into a new tube.
?. [Protocol]
Vortex the antibody-conjugated Nanobeads (to resuspend) at max speed, 5 touches, and prepare the dilutions to test. Add 10µL of pre-dilutedconjugated Nanobeads. Mix well and incubate on ice for 15 minutes. Scale up the volume accordingly if separating more cells. For example, add 100 µL of pre-diluted Nanobeads for separating 1 x 108cells in 1 mL of MojoSort™ Buffer. When working with less than 107cells, use indicated volumes for 107cells.
?. [Protocol]
Wash the cells by adding MojoSort™ Buffer up to 4 mL. Centrifuge the cells at 300xg for 5 minutes.
?. [Protocol]
Discard the supernatant.
?. [Protocol]
Resuspend cells in the appropriate amount of MojoSort™ Buffer and proceed to separation. At least 500 µL is needed for column separation.Note: There are several types of commercially available columns, depending on your application. Choose the one that fits best your experiment:
?. [Data]
Mouse CD19 Nanobeads97.7%94.4%
Mouse CD19 Nanobeads97.7%94.4%
?. [Data]