eamag/protocols-with-steps huggingface dataset:
TITLE: ATP/NADH-enzyme coupled ATPase assay
AUTHORS: Jialin Chen, Marijke De Jaeger, Nathalie Jacobs, Peter Vangheluwe

[DESCRIPTION]
ATP/NADH-enzyme coupled ATPase assay to determine activation of purified ATPase protein via kinetic absorbance measurement. 

[STEPS]
1. Purify the ATPase protein, flash freeze in liquid N2 and store at -80 °C  until use. 

3. Mix the 384-well microplate for 15 s prior kinetic measurement in an absorbance plate reader, set at 25 °C. 

4. Measure absorbance at 340 nm, at 25 °C for30 min to  60 min. This results in at least 10 data points in the linear phase that can be plotted out over time to determine the OD 340 slope reduction.  

2. Use a 384-well clear polystyrene microplate: add 25 µL of the substrate to be tested, together with 40 µL reaction mix, and add 10 µL of 63 millimolar (mM)  ATP at pH 7.0  per well, with a final volume of 75 µL  per well. 

2.1. Prepare serial dilutions of the substrate to be tested, in a final volume of 25 µL  per well. 
If the substrate is dissolved in DMSO, keep the final DMSO concentration in 75 µL  reaction 0.2%. 

2.2. Prepare 40 µL  of reagent mix per well containing: 50 millimolar (mM)MOPS-KOH (pH 7.0 ); 100 millimolar (mM) KCl; 30 millimolar (mM) MgCl2; 2.4 U/µL pyruvate kinase; 2.4 U/µL lactate dehydrogenase; 1.67 millimolar (mM) PEP; and 0.6 millimolar (mM)NADH, in the presence or absence of 600 ng purified ATPase protein. Keep all compounds and the reagent mix at 4 °C .
2.3. Add 10 µL of 63 millimolar (mM) ATP at pH 7.0  per well, and quickly proceed to the acquisition.
TITLE: Immunohistochemical staining of wholemount major pelvic ganglia (MPG) for analysis of myelinated bladder afferents
AUTHORS: Janet R Keast, Peregrine B Osborne, Nicole Wiedmann

[DESCRIPTION]
This protocol describes immunohistochemical procedures applied to wholemount major pelvic ganglia (MPG) for the visualization of myelinated axons filled with cholera toxin subunit B (CTB) and/or neurons transfected with adeno-associated virus (AAV) expressing TdTomato. In this protocol, samples were obtained from rats in which CTB was microinjected into the bladder, and AAV-PHP.S was intravenously administered to preferentially and sparsely label peripheral neurons. In this context, neurofascin (paranode marker) identifies myelinated axons.

[STEPS]
SECTION: Immunohistochemistry
1. Wash whole MPGs in phosphate buffer (PB; 0.1M; pH 7.2) (3 x 30 min)
SECTION: Immunohistochemistry
2. Incubate sections in blocking solution (PB; 10% horse serum; and 0.5% Triton-X) at room temperature for 2 h
SECTION: Immunohistochemistry
3. Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.
SECTION: Immunohistochemistry
4. Wash tissue in PBS (3 x 30 min)
SECTION: Immunohistochemistry
5. Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 24 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.
SECTION: Immunohistochemistry
6. Wash tissue in PBS (3 x 30 min)
SECTION: Immunohistochemistry
7. Mount tissue onto glass slides and coverslip in preferred anti-fade mountant.
TITLE: Mowiol mounting media
AUTHORS: Michael Economo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This makes a low cost mounting media that preserves many fluorescent dyes very well.  It is a non-hardening media.</div><div class = "text-block">This protocol first published in Cold Spring Harbor Protocols.</div></div>

[STEPS]
?. 1.Mix glycerol and Mowiol; dissolve with frequent agitation for 1 h at room temperature.
?. 2.Add H2O. Stir for 1 h at room temperature.
?. 3.Add Tris-Cl. Incubate for 2 h at 50°C with occasional stirring (e.g., for 2 min every 20 min).
?. 4.Centrifuge at 5000g for 15 min.
?. 5.Store 3-mL aliquots of the supernatant for up to 12 mo at -20°C.Mowiol often does not dissolve completely.
TITLE: Validating Responsiveness of AAV-DIO-hM3Dq-DREADD
AUTHORS: Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
To confirm responsiveness of AAV-DIO-hM3Dq-DREADD in DATIRESCre mice. Activation of DREADD receptors in dopamine neurons with CNO should induce a robust increase in locomotion.

[STEPS]
SECTION: Preparation
1. Mice should be at least two-weeks post-surgery.
SECTION: Preparation
2. Habituate mice to wireless running wheels in individually-housed cages.
SECTION: Preparation
2.1. See Victoria Vance, Katerina Rademacher, Ken Nakamura 2023. Behavior Tracking with Running Wheels. protocols.io https://protocols.io/view/behavior-tracking-with-running-wheels-cymrxu56
SECTION: Preparation
3. Prepare CNO for IP injection.
SECTION: Preparation
3.1. 0.5mg/kg CNO dissolved in sterile saline, 500µL volume per mouse.
SECTION: Preparation
3.2. Prepared fresh the day of injection.
SECTION: Procedure
4. Note: All injections should be performed at the same time of day, at least 2–3 hours before the dark cycle.
SECTION: Procedure
5. Day 1: IP inject mice with 500µL of sterile saline.
SECTION: Procedure
6. Day 2: IP inject mice with 500µL CNO.
SECTION: Procedure
7. Day 3 or 4: IP inject mice again with 500µL CNO.
SECTION: Procedure
8. Responders increase their running wheel activity in the 10-60min following IP injection of CNO. Exclude mice that do not respond to IP CNO from further analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is collection of BioLegend protocols for p<span data-sheets-value="{&quot;1&quot;:2,&quot;2&quot;:&quot;Polarization of Mouse CD4+ Cells&quot;}" data-sheets-userformat="{&quot;2&quot;:897,&quot;3&quot;:[null,0],&quot;10&quot;:0,&quot;11&quot;:4,&quot;12&quot;:0}">olarization of mouse CD4+ cells. Please make sure you refer to the correct protocol, depending on whether you are using Th1, Th2, Th17, or Treg.</span></p>

[STEPS]
?.
TITLE: FloodLAMP Inactivation Protocol v3.1
AUTHORS: Randy True

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The inactivation protocol uses a chemical plus heat to break open cells (including virus, if present) and preserve the RNA. The inactivation can be done outside a lab setting, at the point of sample collection or drop-off. This is our preferred mode, and we refer to these as "inactivation stations". Prior to inactivation, the samples may contain live, contagious virus, so it is crucial that personnel use proper PPE and safe handling procedures. Many current community screening efforts are performing non-lab based sample processing (for example, </span><a href="https://dho.pathology.wisc.edu" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Dave O'Connor</span></a><span> and </span><a href="http://masonlab.net" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Chris Mason</span></a><span>), and we have followed their examples with the use of splash guards in addition to PPE.</span></div><div class = "text-block">After inactivation, samples should be refrigerated or stored on ice before same-day processing through the assay.</div><div class = "text-block"><span>On our </span><a href="http://floodlamp.bio" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">website</span></a><span> are our protocols in worksheet form as we use in the lab. This and more information will be live soon.</span></div></div>

[STEPS]
?. [Sample Preparation]
For dry nasal swabs add 2.5mL 1xPBS, soak swabs for 2minutes, remove and discard swabs properly
?. [Inactivation]
Spray all closed tubes with 70% ethanol
?. [Inactivation]
Vortex the 100x Inactivation Solution within five minutes of use
?. [Inactivation]
Add 100x Inactivation Solution to collection tube, add 50uL per 5mL collected sample
?. [Sample Preparation]
For positive controls, spike selected samples with inactivated virions
?. [Inactivation]
For each sample, vortex for 5 seconds, shake and then vortex for another 5 seconds
?. [Inactivation]
Heat the samples at 95C for 8 minutes, be sure to add the QC tube(s) for in situ temperature check
?. [Inactivation]
Chill samples using the frozen lab armor for 4 minutes, alteratively on ice
?. [Inactivation]
Check caps are tight, then Centrifuge samples at 5K rpm for 4 minutes
?. [Inactivation]
Aspirate the clarified samples into new tubes, be sure not to disturb any solids at the bottom of the original tube
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Premature ovarian failure (POF) is a refractory disease; one of the most important goals of treatment is to improve fertility. In this study, umbilical cord mesenchymal stem cells on a collagen scaffold (collagen/UC-MSCs) transplanted into the ovaries of POF mice preserved ovarian function, as evidenced by increased estradiol (E<sub>2</sub>) and anti-Mullerian hormone (AMH) levels, increased ovarian volume, and an increased number of antral follicles. Immunohistochemistry results of Ki67 and Cleaved-Caspase3 indicated transplantation of collagen/UC-MSCs promoted granulosa cell proliferation, which is crucial to oocyte maturation and follicular development. In summary, collagen/UC-MSCs transplantation may provide an effective therapeutic strategy for the treatment of POF.</p>

[STEPS]
TITLE: COREQ (COnsolidated criteria for REporting Qualitative research) Checklist
AUTHORS: pdgalindo, Juan Jesus Torres-Gordillo, Javier Rodríguez-Santero

[DESCRIPTION]
COREQ (COnsolidated criteria for REporting Qualitative research) Checklist, associated with article Parent-school-community relationship: A comparative study of highly effective schools and schools with low effectiveness in Andalusia, by Pablo Delgado-Galindo, Juan-Jesús Torres-Gordillo & Javier Rodríguez-Santero.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol can be used :</p>
<ul>
<li>to isolate novel cultures from natural samples</li>
<li>to isolate novel cultures from enriched samples</li>
<li>to purify existing cultures and remove contaminants</li>
<li>to obtain clonal cultures from a unialgal strain</li>
</ul>
<p> </p>
<p>Reference : Marie, D., Le Gall, F., Edern, R., Gourvil, P. &amp; Vaulot, D. 2017. Improvement of phytoplankton culture isolation using single cell sorting by flow cytometry. J. Phycol. in press.</p>

[STEPS]
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TITLE: High-Throughput Beta-glucuronidase (GUS) assay for Phaeodactylum tricornutum
AUTHORS: Erin Garza, Vincent Bielinski

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>A high-throughput method for measuring β-glucuronidase (GUS) activity in the diatom </span><span style = "font-style:italic;">Phaeodactylum tricornutum</span><span>. This protocol has been optimized for 250 μl volumes.  For larger volumes see the following protocol </span><a href="https://dx.doi.org/10.17504/protocols.io.hefb3bn" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.hefb3bn</span></a><span style = ":UNDERLINE;">, </span><span>which this protocol was based off of.</span></div></div>

[STEPS]
?. [Centrifuge]
Transfer 250 μl of each P. tricornutum culture to a 96-well plate and centrifuge at 3000 x g for 10 min. Discard supernatant.
?. [Lyse]
To lyse the cells, add 150 µl bacterial protein extraction reagent (B-PER, ThermoFisher) to each well and mix by pipetting.
?. [Centrifuge]
Centrifuge plate for 10 min at 3000 x g.  Transfer supernatants to a new 96-well plate, being careful not to disturb the cell debris.
?. [Extract]
Transfer 50 µl of each lysate to a new plate and add 125 µl GUS extraction buffer + 1 mM MUG to each well.  Incubate the plate for 1 h at 37°C.GUS extraction buffer= 50 mM NaPO4H2 (pH 7), 0.1% Triton X-100, and 10 µM βME
?. [Stop Reaction]
To stop the reaction, add 150 µl GUS stop buffer (0.2 M Na2CO3) to each well and mix by pipetting.
?. [Stop Reaction]
Transfer 200 µl quenched reaction to an opaque 96-well plate.
?. [Read fluorescence]
Determine fluorescence using a plate reader. Settings: excitation- 360 nm; emission- 440 nm.
?. [Read fluorescence]
If fluorescence readings are too high to get a readout, dilute with additional stop buffer.
?. [Normalization]
Use remaining cell lysates to perform a BCA assay (or an equivalent assay) to normalize the GUS activity to total cell protein for each culture.
TITLE: Cutting FFPE Sections for Imaging Mass Cytometry
AUTHORS: Marda Jorgensen, Michelle Daniel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">FFPE block are cut into thin sections. </div></div>

[STEPS]
?. [Cooling the FFPE Block]
1.1 Cool down the FFPE block        a. Switch on the cooling device and set it to 4°C        b. Put the FFPE block into the cooling device.
?. [Switching on the microtome]
1.2 Prepare the microtome        a. Connect the cooling unit in the back with the cable and switch it on at thefront.        b. Switch on the microtome using the button at the back of the machine.        c. Install the water bath with the filter.        d. Add deionised water to the water bath and lower the compartment until thegrid is slightly covered with water.        e. Place a fresh blade in the blade holder.        f. Check the water flow. If water is not flowing rinse the tubing with a syringefilled with water.        g. Set the water flow to 3 or 4.        h. Fix the precooled FFPE block to the specimen clamp holder.        i. Adjust the angle between blade and FFPE block by unlocking the clampinglever and then adjusting the orienting screws accordingly.        j. Set the trimming (Trim) and cutting (Feed) thickness by pressing the top knobonce or twice, respectively.        k. Unlock the wheel with the mechanical hand wheel brake on the right-handside underneath the wheel.1.3 Switch on the stretching table        a. Switch it on and set it to 37°C.
?. [Cutting]
1.4 Trimming        a. Always use a new sharp blade.        b. Trim carefully and make sure that your cutting angle is fine, if needed readjust.        c. As soon as you think you are cutting tissue, take up a cut and have a lookunder the microscope.        d. Exchange the water bath before starting cutting sections that you intend tocollect.1.5 Cutting        a. Cut in a uniform and gentle rotation.        b. Let the cut slide down into the water bath.        c. Wait until the cut is flattened before taking it up on a glass slide.        d. Wipe the glass slide dry on the bottom side before placing it on top of thestretcher.        e. Exchange water on a regular basis if sample produces a lot of paraffin debrisand between samples.
?. [Switching off]
1.6 Switching off the microtome        a. Block the wheel to avoid any injuries as soon as you are done cutting        b. Dispose of the blade        c. Stop the water flow by turning the wheel down to 0.        d. Discard the water, clean the water bath, filter and grid, place them to dry.        e. Clean the water slide.        f. Clean the microtome from paraffin debris.        g. Switch off the microtome at the back and disconnect the cooling block.        h. Switch off the FFPE Block cooling device.
?. [Section drying and storage]
1.7 Drying sections        a. Dry the sections overnight or for 1-2 hours at 37°C. Do not extend the dryingfor too long.1.8 Storage of FFPE sections        a. Store all FFPE fresh cut and dried sections at -80°C or stain them within a week.
TITLE: Isolating Fusarium from plant material
AUTHORS: Jenna Moore

[DESCRIPTION]
Protocol for isolating Fusarium (SDS) from soybean plants. A fresh sample is best. Incubating the root sample at 5-10 degrees Celsius encourages sporulation. Samples should be processes no later than 10 days after receiving for best isolation results. Once a pure culture is achieved, find next protocols for single spore culture.  

[STEPS]
SECTION: Isolating Fusarium from plant material
6.1.  After initial observations are recorded, use pruners to cut and discard aboveground plant material (keep material from soil-line down). 
SECTION: Isolating Fusarium from plant material
1. Wash root system. 
SECTION: Isolating Fusarium from plant material
6. Observe growth after 7200 min days and hyphal tip transfer colonies onto fresh APDA. Note: F. virguliforme is slow growing. 

SECTION: Isolating Fusarium from plant material
5. Plates 4-5 root sections onto APDA (with antibiotics). Incubate at room temperature. 25 Room temperature 

SECTION: Isolating Fusarium from plant material
4. Place on sterile paper towels until dried (approximately5 min). 

SECTION: Isolating Fusarium from plant material
2. Using pruners, remove small sections of the taproot and place into sterile petri dish. If necessary, use a scalpel or blade to cut sections into smaller pieces. 
SECTION: Isolating Fusarium from plant material
3. Rinse root sections in 20% bleach for 1 min, followed by sterile water for 1 min
TITLE: Orexin A Analysis in Plasma
AUTHORS: daniel.dautan daniel, Per Svenningsson, Wojciech Paslawski

[DESCRIPTION]
Mouse plasma analysis of Orexin A using Novus Biologicals ELISA kit (NBP2-80231).

[STEPS]
1. Centrifuge samples for 15 min at 1000×g at 4 °C. 

2. Add50 µL of standards' working solutions and samples to 96-well plate in duplicates. 

3. Add 50 µL of biotinylated detection antibody working solution to each well. Cover with the provided plate sealer and incubate for 45 min at 37 °C. 

4. Decant the solution from each well. Add350 µL of wash buffer added to each well and allowed to soak for 1 min. 

5. Decant the solution from each well and pat dry against clean absorbent paper. Repeat this wash step (steps 4-5) 3 times. 
6. Add 100 µLof HRP conjugate working solution to each well. Cover plate with the sealer and incubate for 30 min at 37 °C. 

7. The wash step was performed as described above. Add 90 µL of substrate reagent to each well. Incubate for 15 min at 37 °Cprotected from light. 

8. Add 50 µL of stop solution to each well. Measure the optical density using a micro-plate reader
with absorbance set to 450 nm.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Yeast is a well-characterized genome due to its small size and historical significance in genetics.  The website http://yeastgenome.org/ is a dedicated resource for yeast genomics.</p>
<p> </p>
<p>For this exercise, I want you to create a "Makefile" that will execute this entire pipeline if I type "make."  </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol shows how to dissect the Extensor Digitorum Longus (EDL) muscle and Soleus muscle from a mouse leg. </p>

[BEFORE_START]
<p><strong>Prepare Ringer solution: </strong></p>
<table>
<tbody>
<tr>
<td>
<p><strong>Reagent </strong></p>
</td>
<td>
<p><strong>Concentration </strong></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">NaCl</span></p>
</td>
<td>
<p><span style="font-weight: 400;">145 mM </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">KCl</span></p>
</td>
<td>
<p><span style="font-weight: 400;">5 mM</span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">CaCl</span><span style="font-weight: 400;">2</span></p>
</td>
<td>
<p><span style="font-weight: 400;">2,5 mM</span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">HEPES</span></p>
</td>
<td>
<p><span style="font-weight: 400;">10 mM </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">MgCl</span><span style="font-weight: 400;">2</span></p>
</td>
<td>
<p><span style="font-weight: 400;">1 mM </span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">Glucose</span></p>
</td>
<td>
<p><span style="font-weight: 400;">10 mM</span></p>
</td>
</tr>
<tr>
<td>
<p><span style="font-weight: 400;">pH 7.4 with 1M NaOH</span></p>
</td>
<td>
<p><span style="font-weight: 400;">330 mosmol</span></p>
</td>
</tr>
</tbody>
</table>
<p><strong> </strong></p>
<p><strong>Prepare 3.7% PFA/DPBS:</strong></p>
<p>- Work under a fume hood! </p>
<p>- Use a magnetic stirrer and warm up to 55 °C</p>
<p>- Add a few drops of NaOH for better solubility </p>
<p>- Make sure all of the PFA is dissolved </p>
<p>- Adjust pH to ~7 </p>
<p>- Freeze in portions till use </p>
<p> </p>
<p><strong>Additional materials: </strong></p>
<p>- Dissecting instruments</p>
<p>- Dissecting dishes</p>
<p>- Dissecting needles</p>
<p>- Stereomicroscope </p>
<p>- Parafilm</p>
<p> </p>

[GUIDELINES]
<p style="text-align: justify;"><span style="font-weight: 400;">Two muscles are dissected from a mouse leg: <em>Musculus Soleus</em> and <em>Musculus Extensor Digitorum Longus</em></span><span style="font-weight: 400;"> (</span><span style="font-weight: 400;">EDL). The dissecting process takes place in Ringer solution. The pictures show the right leg, so for the left leg.</span></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><span style="font-weight: 400;">Dissected legs of sacrificed mouse need to be stored in Ringer solution right after dissection and the tube containing the legs should be stored on ice at all times. This is very important because it helps to balance the water and electrolytes and stabilizes the pH value in the tissue. </span></p>
<p style="text-align: justify;"><span style="font-weight: 400;"> </span></p>

[STEPS]
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TITLE: Superoxide Radical-scavenging Activity
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The superoxide radical scavenging effects were examined [11]. Briefly, extract (1 ml) was added to 1 ml of 50 μM NBT solution and 1 ml of 468 μMNADH, and a 1ml aliquot of 60 μM PMS reaction mixture. After 5min, absorbance was read at 560 nm. The inhibition percent and IC</span><span style = "vertical-align:sub;">50</span><span> valueswere calculated.</span></div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Carbapenemase producing Enterobacteriaceae (CPE) are becoming a global healthcare concern. Current laboratory methods for the detection of CPE include screening followed by confirmatory phenotypic and genotypic tests. These processes would generally take >/= 72 hours, which could negatively impact patient care and Infection Control practices. To this end, we developed a protocol for rapid resistance testing (RRT) to detect hydrolysis in a panel of beta lactam antibiotics consisiting of ampicillin, cefazolin, cefotaxime and imipenem, using liquid chromatography tandem mass spectrometry. Ninety-nine beta lactamase producing Enterobacteriaceae isolates were used to evaluate the RRT method, 54 isolates were CPE and 45 isolates were Class A or AmpC beta lactamase producing Enterobacteriaceae but not carbapenemase producers. We also tested 10 E.coli isolates that were susceptible to ampicillin, cefazolin, cefotaxime and imipenem. Receiver Operating Characteristic (ROC) Curves analysis showed that imipenem had a sensitivity and a specificity of 100% for carbapenemase detection at hydrolysis cut off values that are greater than 50% and less than or equal to 80%. The RRT protocol can be conducted in a time frame of less than 2 hours. This preliminary study shows that the rapid resistance testing protocol might have utility for the rapid detection of CPE. Additional work with a greater number and a variety of beta-lactamase producing Enterobacteriaceae isolates is required to validate these preliminary findings.

[STEPS]
?.