eamag/protocols-with-steps huggingface dataset:
TITLE: Sandwich ELISA for the quantification of alpha-synuclein
AUTHORS: Nathalie Lengacher, Brit Mollenhauer, Michael G. Schlossmacher

[DESCRIPTION]
This protocol details the procedure to quantify total alpha-synuclein from tissue and biological fluids by ELISA.

[STEPS]
SECTION: Sandwich ELISA for alpha-synuclein
1. 
 

SECTION: Coating - Day 1
2. Coat 384-well plate with primary antibody.
SECTION: Coating - Day 1
2.1. Make Coating buffer  200 millimolar (mM)   NaHCO3 with 0.02% sodium azide (pH 9.6).

SECTION: Coating - Day 1
2.2. 1 0 antibody most used is syn1 at 1:500 dilution (BD 610787).
SECTION: Coating - Day 1
3. Add 25 µL per well (avoiding outer wells).

SECTION: Coating - Day 1
4. Incubate on rocker at  4 °C   .

SECTION: Blocking - Day 2
5.  Make blocking buffer (PBS+2.5% gelatin, 0.025% tween).
SECTION: Blocking - Day 2
6. Wash plate 3X using 1xPBS-T in plate washer.
SECTION: Blocking - Day 2
7. Blot on paper towel.
SECTION: Blocking - Day 2
8. Add 80 µL per well of blocking buffer.

SECTION: Blocking - Day 2
9. Incubate at 37 °C for 60 min.

SECTION: Blocking - Day 2
10. During incubation, prepare standards and samples on ice, dilute in blocking buffer (BB).

SECTION: Samples-  Day 2
11. Standards – use full length recombinant alpha-synuclein. Stock aliquoted upon opening.
SECTION: Samples-  Day 2
11.1. Make serial dilutions in blocking buffer, starting from 10 1792 stock.

SECTION: Samples-  Day 2
12. Dilute samples in blocking buffer.
SECTION: Samples-  Day 2
13. Wash plate 3X, blot on paper towel.
SECTION: Samples-  Day 2
14.  Add 25 µL per well of standards and samples (in duplicate).

SECTION: Samples-  Day 2
15.  Incubate at Room temperature for 120 min.

SECTION: Detection antibody - Day 2
16. Bring blocking buffer to Room temperature before use.

SECTION: Detection antibody - Day 2
17. Dilute detection antibody in blocking buffer.
2 o antibody most used is biotinylated-hSA4, at 1:200 dilution (commercially MJFR1 ab138501; biotinylated in-lab).
SECTION: Detection antibody - Day 2
18. Wash plate 3X, blot on paper towel.
SECTION: Detection antibody - Day 2
19. Add 25 µL per well of detection antibody.

SECTION: Detection antibody - Day 2
20. Incubate at 37 °C for 60 min.

SECTION: Avidin-alkaline phosphatase - Day 2
21. Wash plate 5X, blot on paper towel.
SECTION: Avidin-alkaline phosphatase - Day 2
22. Use avidin-alkaline phosphatase at 0.6 1824 of blocking buffer.

SECTION: Avidin-alkaline phosphatase - Day 2
23. Add 25 µL per well of AAP.

SECTION: Avidin-alkaline phosphatase - Day 2
24. Incubate at 37 °C  for  30 min.

SECTION: Substrate - Day 2
25. Make pNPP - 1 of each tablet for 5 mL ddH2O (cover tube in aluminum, rock to dissolve).

SECTION: Substrate - Day 2
26. Wash plate 5X, blot on paper towel.
SECTION: Substrate - Day 2
27. Add 25 µL/well of pNPP, read plate immediately.

SECTION: Substrate - Day 2
27.1. Read absorbance at 405nm, every 2.5 minutes for 60 min.

SECTION: Substrate - Day 2
27.2. Use data from a read when the standards are linear.
TITLE: CODEX - Poly-l-Lysine Cover-Slip Preparation
AUTHORS: Leigh Propper, Franchesca Farris, Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Poly-Lysine Coverslip Preparation </div><div class = "text-block">This protocol describes the process of creating Poly-lysine-coated coverslips that are required for mounting tissue sections for the CODEX® experiment workflow.</div></div>

[STEPS]
?. Remove 12-15 cover-slips from box. Cover-Slip brand and type are REQUIRED, not suggested.
?. Gently place the desired amount of cover-slips into the beaker, spreading them out and allowing both surfaces to have contact with the solution.
?. Slowly swirl the beaker to spread the cover-slips around the base.
?. Add 7 mL of poly-lysine solution above the cover-slips to ensure that all are fully covered.
[Poly-Lysine]
?. Cover the beaker with plastic wrap or parafilm, and secure tightly with a rubber band to prevent evaporation.
?. Leave cover-slips in poly-lysine solution for a minimum of 12 hours and up to one week at room temperature.
[or up to one week.]
?. After waiting the required incubation period, carefully remove the rubber band and parafilm from around the neck of the beaker.
?. Gradually pour the remaining poly-lysine solution into the proper waste container.
?. Fill the same beaker containing the cover-slips to half volume with double-distilled water(ddH20) or purified water.
?. Swirl the contents to mix the solution.
?. Let the beaker and cover-slips sit for 30 seconds. To prevent removal of poly-lysine, do not soak in water for >1 minute during each washing step.
?. Slowly pour off the water into the sink. This completes Wash #1
?. [ and repeat steps 9-12 ( Wash #2)]
?. [ and repeat steps 9-12 ( Wash #3)]
?. [ and repeat steps 9-12 ( Wash #4)]
?. [ and repeat steps 9-12 ( Wash #5)]
?. [ and repeat steps 9-12 ( Wash #6)]
?. [ and repeat steps 9-12 ( Wash #7)]
?. After completing the 7 washes, place 2 lint-free Wypall towels on the bench top.
?. Remove the cover-slips from the water, placing them on top of the first set of towels.Ensure the cover-slips are not overlapping to allow proper drying.
?. Using the specialty forceps indicated for use by CODEX,flip over each cover-slip onto the second clean towel to dry the reverse side.
?. Leave the cover-slips on the Wypall towel to dry.
?. When the cover-slips are dry, the Poly-Lysine-coated cover-slips can be stored in a sterile petri dish or similarly covered container for up to 2 (two) months.
?. Pour enough poly-l-lysine solution to cover the bottom of the clean beaker.
TITLE:  Preparation of biological monolayers for producing high-resolution scanning electron micrographs Code: PLOS2021 [PONE-D-21-26669] - [EMID:f8569bc7b537f6e4]
AUTHORS: Shireen Mentor, Franscious Cummings, David Fisher, shireen.mentor

[DESCRIPTION]
Scanning electron microscopy (SEM) provides a technical platform for nanoscopic mapping of biological structures. Correct preparation of SEM samples can provide an unprecedented understanding of the nexus between cellular morphology and topography. This comparative study critically examines two coating methods for preparing biological samples for scanning electron microscopy, while also providing novel advice on how to prepare in vitro epithelial or endothelial samples for high-resolution scanning-electron microscopy (HR-SEM). Two obstacles often confront the biologist when investigating cellular structures grown under tissue culture conditions, viz., how to prepare and present the biological samples to the HR-SEM microscope without affecting topographical membrane and cellular structural alterations. Firstly, our use of the Millicell cellulose inserts on which to grow our cellular samples in preparation for HR-SEM is both novel and advantageous to comparing the permeability function of cells to their morphological function. Secondly, biological material is often non-conducting, thermally sensitive and fragile and, therefore, needs to be fixed correctly and coated with thin conducting metal to ensure high-resolution detail of samples. Immortalized mouse brain endothelial cells (bEnd5) was used as a basis for describing the preferences in the use of the protocol. We compare two biological sample coating modalities for the visualizing and analysis of texturized, topographical, membranous ultrastructures of brain endothelial cell (BEC) confluent monolayers, namely, carbon and gold:palladium (Au:Pd) sputter coating in preparation for HR-SEM. BEC monolayers sputter-coated with these two modalities produced three-dimensional micrographs which have distinctly different topographical detail from which the nanostructural cellular data can be examined. The two coating methods display differences in the amount of nanoscopic detail that could be resolved in the nanosized membrane cytoarchitecture of BEC monolayers. The micrographical data clearly showed that Au:Pd sputter-coated samples generate descript imagery, providing useful information for profiling membrane nanostructures compared to carbon-coated samples. The recommendations regarding the contrast in two modalities would provide the necessary guidance to biological microscopists in preparing tissue culture samples for HR-SEM.

[STEPS]
TITLE: Crystallization of Enterovirus coxsackievirus A16 2A protease
AUTHORS: ryan Lithgo, Peter Marples, Lizbé Koekemoer, Daren Fearon

[DESCRIPTION]
Picornaviridae coxsackievirus A16 is the causative agent of paediatric hand-foot-and-mouth disease, and a target for pandemic preparedness due to the risk of higher order complications in a large-scale outbreak. The 2A protease of the virus is responsible for self-cleavage from the poly protein, allowing for correct folding and assembly of capsid proteins in the final stages of viral replication. Inhibition deranges capsid folding and assembly, preventing formation of mature virions in host cells and making the protease a valuable target for antiviral activity. This protocol was used to grow coxsackievirus A16 crystals that were applied high-throughput crystallographic fragment screening on the target.

[STEPS]
SECTION: Crystallization experiment
3. Protein and buffer requirements:
43.2 µL20 mg/mL 
3.36 mL 
14.4 µL   seeds, dilution 1:1000
SECTION: Crystallization experiment
5. Dispense 35 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 150 nL20 mg/mL  to each lens using the SPT mosquito.
Dispense  150 nL to each lens using the SPT mosquito.
Dispense  50 nL to each lens using the SPT mosquito.

Drop ratio: 3:3:1 ratio (150 nl   : 150 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 nl
SECTION: Crystallization experiment
4. Crystallisation screen composition:
13.5 % PEG 20000
0.1 Molarity (M)  MES pH 6.7 


Stock solutions used:
1 Molarity (M)  MES pH 6.7  
50% w/v PEG 20000

 

SECTION: Crystallization experiment
7. Crystal typically form after ~24hrs
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12hrs and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
 
P100 8 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
2. Prepare seed stock:
 
1: 1000 dilution    seeds
SECTION: Data collection at Synchrotron
8. Diamond Light Source
Unattended Data Collection (UDC)
Data Collection Temperature: 100K
Detector: DECTRIS EIGER2 X 9M
Beamline: I04-1
Wavelength:  0.9212 Å
Resolution (Å): 1.21
Beam Size (μm): 60 X 50
Number of images: 3600
Oscillation: 0.10°
Exposure (s): 0.0020
Transmission (%): 100
Flux (ph/s): 9.50e+11
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>Primers:<br /></strong>CPS1.1 primer A: GTA GWA TWT TYT AYA TTG AYG TWG G<br />CPS8.1 primer B: ART AYT TDC CDA YRW AWG GWT C<br /><br /><strong>Reaction Mix:<br /></strong></p>
<table style="width: 400px; height: 200px;">
<tbody>
<tr>
<td>Water</td>
<td>14.25&mu;l</td>
</tr>
<tr>
<td>10x LA Buffer</td>
<td>2.5&mu;l</td>
</tr>
<tr>
<td>dNTP mix, 2.mM each</td>
<td>5.0&mu;l</td>
</tr>
<tr>
<td>Primer A, 100&mu;M</td>
<td>1.0&mu;l</td>
</tr>
<tr>
<td>Primer B, 100&mu;M</td>
<td>1.0&mu;l</td>
</tr>
<tr>
<td>LA Takara HS 2.5U/&mu;l</td>
<td>0.25&mu;l</td>
</tr>
<tr>
<td>DNA (viral lysates)</td>
<td>1.0&mu;l</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>Total = 25&mu;l</td>
</tr>
</tbody>
</table>
<br />
<p><strong>Cycling<br /></strong></p>
<table style="width: 350px; height: 150px;">
<tbody>
<tr>
<td>94<sup>o</sup>C</td>
<td>3 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>94<sup>o</sup>C</td>
<td>15 sec</td>
<td rowspan="3">&nbsp;30 cycles&nbsp;&nbsp;</td>
</tr>
<tr>
<td>35<sup>o</sup>C</td>
<td>1 min, ramped up 0.3<sup>o</sup>C/cycle</td>
</tr>
<tr>
<td>73<sup>o</sup>C</td>
<td>1 min</td>
</tr>
<tr>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>73<sup>o</sup>C</td>
<td>10 min</td>
<td>&nbsp;</td>
</tr>
<tr>
<td>4<sup>o</sup>C</td>
<td>Hold</td>
<td>&nbsp;</td>
</tr>
</tbody>
</table>
<p><strong>&nbsp;</strong></p>
<p>Products in the range of 600 bp (may also see less intense band ~400 bp)</p>
<p><strong>&nbsp;</strong></p>

[STEPS]
TITLE: Preparation of 100mg/ml kanamycin solution
AUTHORS: Jenny  Molloy, Nadine    Mowoh

[DESCRIPTION]
Kanamycin is an aminoglycoside antibiotic which binds the 30S ribosomal subunit; causes mis-translation. It is used for the selection of cells containing Kanamycin-resistant plasmids on plates and on liquid media at a working concentration of 50-100 µg/mL. 

It is most often prepared as a 1000x working stock of 50 -100 mg/mL that is stored at -20 ºC. 

The kanamycin solution is measured and then mixed into a container of liquid or plate media before it has solidified. Cells are then streaked onto the plates or grown in the liquid media. Only cells containing the plasmids resistant to kanamycin will grow.

[STEPS]
SECTION: Preparing 1000x kanamycin sulphate stock solution
1. Clean work surfaces and work space using freshly prepared 10 % (v/v) bleach, followed by 70 % (v/v) alcohol.

SECTION: Preparing 1000x kanamycin sulphate stock solution
2. Using the electronic balance, carefully weigh out 0.4 g of kanamycin sulphate powder (CAS#: 70560-51-9; Linear Formula: C18H36-37N4-5O10-11 · H2SO4) into an appropriate size weighing boat using a spatula. 

 

SECTION: Preparing 1000x kanamycin sulphate stock solution
3. Carefully tip the powder into a sterile 50 mL centrifuge tube.

SECTION: Preparing 1000x kanamycin sulphate stock solution
4. Using a 10 mL measuring cylinder, measure out 4 mL of sterile distilled or deionised water.

SECTION: Preparing 1000x kanamycin sulphate stock solution
5. Pour 2 mL water into the 50 mL centrifuge tube containing the kanamycin powder.

SECTION: Preparing 1000x kanamycin sulphate stock solution
6. Use the remaining 2 mL to rinse the weighing boat and pouring that into the 15 mL falcon tube to make 4 mL total and mix gently by inversion.

SECTION: Preparing 1000x kanamycin sulphate stock solution
7. Using the sterile 20 mLsyringe, aspirate the solution from the falcon tube

SECTION: Preparing 1000x kanamycin sulphate stock solution
8. Affix a sterile 0.22 µm micro filter to the end of the syringe and dispense thee solution into a new sterile  15 mL centrifuge tube.
SECTION: Preparing 1000x kanamycin sulphate stock solution
9. Pipette 1000 µL of the filtered 100 mg/mL Kanamycin solution into 4 x sterile 1.5 mL microcentrifuge tubes.

SECTION: Preparing 1000x kanamycin sulphate stock solution
10. Close the tubes, label and store them at-20 °C if they are not to be used immediately. This is a 1000x stock of 100 µg/µL Kanamycin which is the normal working concentration in liquid or solid media. The stock can be diluted before use to reach the desired concentration.
TITLE: Robust isolation protocol for mouse leukocytes from blood and liver resident cells for immunology research
AUTHORS: Dorien De Pooter, Ben De Clerck, Koen Dockx, Domenica De Santis, Sarah Sauviller, Pascale Dehertogh, Matthias Beyens, Isabelle Bergiers, Isabel Nájera, Ellen Van Gulck, Nádia Conceição-Neto, Wim Pierson

[DESCRIPTION]
Research on liver-related conditions requires a robust and efficient method to purify viable hepatocytes, lymphocytes and all other liver resident cells, such as Kupffer or liver sinusoidal endothelial cells. Here we describe a novel purification method using liver enzymatic digestion, followed by a downstream optimized purification. Using this enzymatic digestion protocol, the resident liver cells as well as viable hepatocytes could be captured, compared to the classical mechanical liver disruption method. Moreover, single-cell RNA-sequencing demonstrated higher quality lymphocyte data in downstream analyses after the liver enzymatic digestion, allowing for studying of immunological responses or changes. In order to also understand the peripheral immune landscape, a protocol for lymphocyte purification from mouse systemic whole blood was optimized, allowing for efficient removal of red blood cells. The combination of microbeads and mRNA blockers allowed for a clean blood sample, enabling robust single-cell RNA-sequencing data. These two protocols for blood and liver provide important new methodologies for liver-related studies such as NASH, hepatitis virus infections or cancer research but also for immunology where high-quality cells are indispensable for further downstream assays.

[STEPS]
TITLE: Cleaning Microscopes (Covid-19 Shutdown Re-Entry, Srivastava Lab)
AUTHORS: Srivastava Lab

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is based on: </span><a href="https://www.gerbi-gmb.de/sites/default/files/2020-03/Recommendations_SARS-CoV-2_GerBI-GMB_2020-03-18.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">Recommendations for operating Core Facilities in a research environment during the SARS-CoV-2 pandemic:</span></a></div><div class = "text-block"><span style = "font-weight:bold;">Disclaimer: </span><span>The SOP presented here has been designed by the Srivastava Lab at Harvard University and is being shared outside of Harvard for informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that you do so at your own risk; you should ensure that any local guidance is also adhered to. None of the authors, contributors, administrators, or anyone else associated with </span><a href="http://protocols.io" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">protocols.io</span></a><span>, can be held responsible for your use of the information contained in or linked to from these web pages.</span></div></div>

[STEPS]
?. When using the microscope and adjacent computer, always wear gloves
?. Wipe hard surfaces (knobs, body of scope, screens) with 70% ethanol before use
?. Place a new piece of plastic wrap over oculars before use
cleaning oculars too frequently can cause the rubber to degrade
?. When done, dispose of plastic wrap and wipe hard surfaces with 70% ethanol
TITLE: 50 mM Phosphate Buffer (with 0.02% sodium azide option)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">50 mM Phosphate Buffer can be used as a general buffer solution for multiple applications.  It is suitable for use as a buffer for specimen embedding, as part of brain preparation associated with TissueCyte 2P Serial Imaging, described in PF0315 TissueCyte Specimen Embedding Acrylamide Coembedding.</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: Protocol collection: Sampling and characterizing cheese rind microbial communities
AUTHORS: Adair  Borges, Emily C.P. Weiss

[DESCRIPTION]
Cheese rind microbiomes are a validated and stable experimental platform for microbial community research. These communities are relatively simple, reproducible, and easy to grow in the lab. This set of protocols describes how to collect and store cheese rind microbiome samples for isolation of pure cultures, metagenomic sequencing, and other downstream applications. This collection also includes specific protocols for virome extraction and for the isolation of full community and viral-only DNA from cheese samples.

[STEPS]
TITLE: Preparo de Meio L15
AUTHORS: Fabio Gomes

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocolo de Manutenção de Infecção (DENV e ZIKV) de Células C6/36 - PLO</div></div>

[STEPS]
?. [Preparo Meio L15]
Diluir 29g de triptose em 1L
?. [Preparo Meio L15]
Autoclavar e armazenar.
?. [Preparo Meio L15]
Diluir 0.75g de bicarbonato de sódio, 0.20g de L-glutaminaColocar em 800ml de água
?. [Preparo Meio L15]
Ajustar o ph 7.4
?. [Preparo Meio L15]
Acrescentar ao meio 100ml da triptose autoclavada
?. [Preparo Meio L15]
Avolumar para 1 L
?. [Preparo Meio L15]
Filtrar e estocar na geladeira
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This mixture is used with <a href="https://www.protocols.io/view/Cesium-Chloride-Dialysis-for-Viruses-c7jzkm" target="_blank">Cesium Chloride Dialysis for Viruses</a>

[GUIDELINES]
Needed:<br />- 3M NaCl<br />- 0.1M Tris-Cl, pH 7.5<br />- 0.1M MgCl2

[STEPS]
?. 
?. 
?. 
?.
TITLE: Coating coverslips for cell culture
AUTHORS: Chuyu Chen, Ciarra Smith

[DESCRIPTION]
Coated cover-slips provide a nourishing adherent surface for cell culture. This protocol provides step by step instruction on how to coat coverslips for cell culture. 

[STEPS]
SECTION: Poly L-Lysine/ Poly D-Lysine coating
1. Precoat the coverslips with poly-L-lysine (P5899) or poly-D-lysine (P7280) at 100 μg/ml.
SECTION: Poly L-Lysine/ Poly D-Lysine coating
2. This is incubated for 30 minutes or more at room temperature. The coverslips are washed with sterile water and allowed to dry.
SECTION: Poly L-Lysine/ Poly D-Lysine coating
3. They can be stored at room temperature until ready to use (up to 1 month). 
SECTION: Day before culturing: Laminin coating
5. Incubate overnight in a humidified 37°C, 5% CO2 incubator or at 37 °C for 30 minutes.
SECTION: Day before culturing: Laminin coating
6. Remove excess laminin and rinse with PBS twice. Alternatively, wash with DME (50 μL) twice.
SECTION: Day before culturing: Laminin coating
7. Add the cells to the coverslips/slides and culture as needed.
SECTION: Day before culturing: Laminin coating
4. Coverslips are then coated with 30 - 50 μl of laminin (50 μg/ml) which has been warmed. This is prepared the day before culturing of cells.
TITLE: Dot mutation
AUTHORS: Zhujun Wei

[STEPS]
?. The entire plasmid was amplified reversely by PCR using primers with the fragment sequence that you want to replace.
?. The temple plasmids in the PCR process were digested by DpnI enzyme.
?. The digested product was transferred into DH5α. Overnight culture them at 37℃.
?. To determine whether the vector was constructed successfully, colony PCR and enzyme digestion were done.
?. Select the positive results for sequencing and the final results were obtained.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Traditionally, social scientists perceived causality as regularity. As a consequence, qualitative comparative case study research was regarded as unsuitable for drawing causal inferences. Apparently, few cases cannot establish regularity. The dominant perception of causality has changed, however. Nowadays, social scientists define and identify causality through the counterfactual effect of a treatment. This brings causal inference in qualitative comparative research back on the agenda since obviously comparative case studies can identify treatment effects. We argue that the validity of causal inferences from the comparative study of cases depends on the employed case-selection algorithm. We employ Monte Carlo techniques to demonstrate that different case-selection rules strongly differ in their ex ante reliability for making valid causal inferences and identify the most and the least reliable case selection rules.</p>

[STEPS]
?. 
?. 
?.