eamag/protocols-with-steps huggingface dataset:
TITLE: Photometry Acquisition in Freely Moving Mice
AUTHORS: Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
This protocol describes the steps for acquiring photometry data for freely moving mice.

[STEPS]
SECTION: Habituation
1. Habituate the mouse to tethering and the behavioral chamber for 10 minutes/day for two days prior to starting testing sessions.
SECTION: Habituation
1.1. Scruff the mouse and attach the optical fiber patch cable to the mouse’s implant.
SECTION: Habituation
1.2. Place mouse in the behavioral chamber (a clear acrylic cylinder, 25 cm in diameter).
SECTION: Habituation
1.3. Monitor the mouse for the duration of the session to ensure it does not become tangled by the patch cable and moves freely about the chamber. Also check that the patch cable does not twist around itself such that the mouse cannot move freely.
SECTION: Computer and Optical Setup
2. Computer and optical setup
SECTION: Computer and Optical Setup
2.1. Turn on the computer.
SECTION: Computer and Optical Setup
2.2. Turn on TDT RZ5P (power button on top left of box, blue light turns on).
SECTION: Computer and Optical Setup
2.3. Flip the red switch on the Photoreceiver (left switch) towards you to turn it on. Do not touch the other switch.
SECTION: Computer and Optical Setup
2.4. Turn on the LED Driver (switch on back of box). You should see the screen turn on. Make sure you are in “External Command” mode and use the click wheel and LED button to select the LED wavelengths you wish to turn on.
SECTION: Computer and Optical Setup
2.5. Open Synapse Software (TDT). Make sure that the desired experimental design is selected. Click “Preview” and serially cover and uncover the sleeve at the end of the patch cable to ensure that the light is emitted properly and the photodetectors are functional.
SECTION: Testing
3. Testing
SECTION: Testing
3.1. Scruff the mouse and attach optical cable to the photometry implant, and place into the behavioral chamber.
SECTION: Testing
3.2. Press the red “Record” button in Synapse and enter the file name for the experiment. This will start the experiment. It is important that you start the photometry recording before any other signals, as it will serve as the master data file, collecting timestamps from all other devices.
SECTION: Testing
3.3. To check the fiber signal, right click the axis on the computer screen and select auto scale to bring signal into frame.
SECTION: Testing
3.4. Recording will end after 10 minutes. Remove mouse from chamber and unplug the mouse from the optical cable. Return to home cage. Before testing additional mice, clean the chamber with 70% ethanol.
SECTION: Cleanup
4. Cleanup
SECTION: Cleanup
4.1. Remove mouse from chamber and unplug optical cable. Return to home cage.
SECTION: Cleanup
4.2. Clean chamber with 70% ethanol between mice and at end of the day.
SECTION: Cleanup
4.3. When experiments are completed for the day, turn off the photodetectors, LED drivers, TDT system, and Synapse programs. Transfer and back up files.
TITLE: Neural Rosette Formation and Selection
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. On Day 5 of neural aggregate formation, remove media (by pipetting) and carefully wash spheres with of pre-warmed DMEM/F12. Repeat 2 times.
100 µl
Do not break apart spheres. Neural spheres are very delicate at this stage. An alternative approach is to remove   of spent media and wash with  . Add  . Transfer   of spheres and media to the new PLO/laminin-coated well. This approach will transfer more dead cells into the new well.
?. Remove the last wash and add  of neural induction media to each well.
50 µl
?. Aspirate laminin from one well of the pre-coated plate. Using 200 μl sterile tips, carefully pipet up spheres from wells using volume and transfer thirty-two spheres per well. Repeat above steps for the remaining wells. Incubate cells in , 5% CO2 and 95% humidified chamber and distribute evenly by making a “T” motion.
100 µl
?. After , examine attached aggregates. Remove medium and replace with 2mls/well fresh neural induction medium daily.
If some aggregates have not attached, carefully pipet out all medium and replace with 1ml/well fresh neural induction medium. Once 90-100% of aggregates attach, exchange medium daily with 2mls/well neural induction medium.
?. Monitor spheres daily under microscope for formation of neural rosette structures. Neural rosettes are ready to harvest when spheres have completely flattened and clusters are clearly visible (3-7 days after plating, line dependent).
?. Harvest neural rosettes by aspirating spent medium. Add of pre-warmed DMEM/F12 to each well to remove unattached cells (repeat if necessary).
1 ml
?. Add  of Neural Rosette Selection reagent to each well and incubate for up to  at (check cells at . Cells are typically collected after 30-45 min incubation. Look for rosette structure to be rounding up without the disturbance of other surrounding cells).
1 ml
?. Carefully remove Neural Rosette Selection reagent with a pipet, being careful not to disturb rosette clusters. Add  DMEM/F12 to each well, then using a p1000 detach rosette clusters by  rinsing over them.
1 ml
?. Transfer rosette material from 1 well into a 15 mL conical tube for cryopreservation of neural rosettes and from 2 wells into a separate 15 mL conical tube for neural progenitor expansion. Do not triturate clusters.
To maintain a pure culture, it is best to leave some rosettes behind rather than collect all of the rosettes and additional cells.
?. Centrifuge rosette clusters at 750 rpm for.
TITLE: SDS-PAGE gel electrophoresis
AUTHORS: Steven J Burgess, Lynn    Doran

[DESCRIPTION]
SDS-PAGE gel electrophoresis protocol for analyzing samples from plant leaf tissue via immunofluorescence. In this protocol no Coomassie blue is added to samples, the reason is that this interferes with the fluorescent signal during immunoblot. Instead, samples have already been prepared in Laemmli buffer (minus coomassie, protein extraction procedure), the leading edge of samples can be visualized due to the presence of chlorophyll.  

Note 
- When using 15 well, 0.75 mm comb, try to limit the volume loaded to 10 μL to minimize the risk of spillover of protein between wells. 
- Ensure that accurate volume is pipetted by removing sample stuck to the outside of the pipette tip by wiping the tip on the rim of the sample tube to remove any residual liquid.


Literature: 
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6040.pdfhttps://www.bio-rad.com/webroot/web/pdf/lsr/literature/10026447.pdf

[BEFORE_START]
Extract protein from samples via Leaf Protein Extraction for Immunoblot  and determine Protein Concentration using Qubit 4 Fluorometer.

Previously extracted and quantified protein samples can be stored at -20 °C.

[STEPS]
SECTION: Prepare gel tank and buffers
1. Create a 1X working dilution of  Tris/Glycine/SDS buffer (~1 L  is required per gel tank) by diluting 10X stock 1:10 with distilled H2O.

SECTION: Prepare gel tank and buffers
2. Carefully remove the comb from the precast gel and the tape across the bottom. 
SECTION: Prepare gel tank and buffers
3. Assemble the Mini-PROTEAN electrophoresis cell and fill the inner chamber with buffer and the outer chamber up to the recommended mark 
 

SECTION: Prepare gel tank and buffers
4. Wash the wells with running buffer by pipetting up and down
 

SECTION: Prepare Samples
5. In fresh microcentrifuge tubes, create a dilution of each sample using 1x PEB to a concentration of 3 μg /uL of total soluble protein. 
 


 

SECTION: Prepare Samples
7. Load 3 µL of Chameleon™ Duo Pre-stained Protein Ladder to the first well.

SECTION: Prepare Samples
8. Load 10 µL of each sample (30 µg of total soluble protein) per lane.

SECTION: Running Gel
9. Run precast gels at 200 V for ~30 min or until the samples have reached the end of the gel. In some applications it may be advantageous to run the chlorophyll off the end of the gel to improve fluorescence and signal on the protein of interest.

 

 

SECTION: Running Gel
10. Carefully open precast gel case using an opening lever, by inserting where the black arrows indicate on the gel case.
SECTION: Running Gel
11. Remove stacking gel with a blade
SECTION: Running Gel
12. Proceed either directly to Protein Transfer using Bio-rad TransBlot Turbo  or  Total Protein Staining.

SECTION: Prepare Samples
6. Briefly vortex the sample to shear any DNA contamination.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Este é um guia de como deverá ser a consulta anestésica para eletroconvulsoterapia.
Nossa equipe exige um padrão mínimo de qualidade destas consultas, que devem incluir informações específicas e muito relevantes para a realização de anestesias com o máximo de segurança no nosso instituto.

Este guia se aplica para consultas por membros internos e externos à equipe de anestesiologia que atua no ICS-Brasília. Caso uma consulta por profissional externo não contenha os critérios mínimos de qualidade, não será considerada como passo realizado para realização de ECT com nossa equipe.

[BEFORE_START]
Leia ao menos uma vez todo o protocolo antes de iniciar a aplicação.

[GUIDELINES]
Sugerimos que este protocolo seja usado apenas por médicos anestesiologistas.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Paraffin sections (4 μm thickness) of formalin-fixed human pancreases were treated with heat/citrate buffer for antigen retrieval. Hpse was detected immunohistochemically using HP130 mouse anti-human Hpse mAb (Insight), with biotinylated anti-mouse IgG (1/250) (PK-2200, Vector Labs) and avidin-biotin-complex (ABC reagent; PK-2200, Vector Labs). Background staining was checked using the corresponding isotype control Ig instead of the primary antibody. 3-amino-9-ethylcarbazole (AEC) was used as the chromogen. Stained sections were imaged using a light microscope with attached camera (Olympus BX41).</p>

[BEFORE_START]
<p>Materials:</p>
<p> </p>
<ol>
<li>Prepare graded alcohols and xylene for deparaffinizing tissue sections: 2 x xylene (250 ml/slide container), 2 x absolute ethanol (250 ml/slide container), 1 x 90% ethanol (250 ml), 1 x 70% ethanol (250 ml).</li>
</ol>
<p> </p>
<ol start="2">
<li>Prepare acetate buffer components:</li>
</ol>
<p>(i) 0.1N acetic acid: 290 μl glacial acetic acid in 50 ml deionized water</p>
<p>(ii) 0.1M sodium acetate: 410 mg anhydrous CH<sub>3</sub>COONa in 50 ml deionized water.</p>
<p>Prepare 0.1M acetate buffer (pH 5.2) by mixing 10.5 ml 0.1N acetic acid and 39.5 ml 0.1M sodium acetate.</p>
<p> </p>
<ol start="3">
<li>Prepare stock solution of 3-amino-9-ethylcarbazole (AEC; chromogen, 8 mg/ml: 40 mg AEC in 5 ml N-N-dimethyl formamide; protect from light and refrigerate at 4°C.</li>
</ol>
<p> </p>
<ol start="4">
<li>Prepare M.O.M. diluent: 200 μl M.O.M. protein concentrate stock solution (M.O.M. Immunodetection kit, PK-2200, Vector) in 2.5 ml phosphate-buffered saline (PBS) for use either as a blocking step to minimize non-specific Ig binding or for diluting antibodies.</li>
</ol>
<p> </p>
<ol start="5">
<li>Mabs and pAbs:</li>
</ol>
<p>HP130 mouse anti-human heparanase mAb, Insight Biopharmaceuticals #INS-26-1-0000-21 (alternative: HPA1 rabbit anti-human heparanase, Insight Biopharmaceuticals #INS-26-2-0000)</p>
<p>Biotinylated anti-mouse IgG, M.O.M immunodetection kit, Vector Labs # PK-2200 (alternative: HRP-swine anti-rabbit Ig, Dako #PO217)</p>
<p>Mouse IgG1<sub>Κ</sub>, eBioscience # 14-4714-85 (alternative Rabbit IgG, Southern Biotech, #0111-01)</p>
<p> </p>
<p> </p>
<ol start="6">
<li>Other reagents:</li>
</ol>
<p>Hydrogen peroxide (30% w/w), Chem-Supply Pty Ltd (Australia) #HA154-500M<br /> Methanol, Merck # CAS-No. 67-56-1</p>
<p>3-Amino-9-ethylcarbazole (AEC), Sigma-Aldrich #A5754</p>
<p>Animal free blocker, Vector Labs #SP-5030</p>
<p>Stock protein concentrate, M.O.M immunodetection kit, Vector Labs # PK-2200</p>
<p>Avidin-biotin complex (ABC), M.O.M immunodetection kit, Vector Labs # PK-2200</p>
<p>N-N-dimethyl formamide, Sigma #D158550</p>
<p>Glycergel mounting medium, Dako #C0563</p>
<p> </p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The following is a general procedure guide for preparation and staining of acetone-fixed <strong>frozen</strong> tissues using a purified, unconjugated primary antibody, biotinylated secondary antibody and streptavidin-horseradish peroxidase (Sav-HRP) and DAB detection system. Because each antigen differs in terms of requirement for fixation, amplification step, etc., it is not possible to write an inclusive protocol that will work for all antigens. The user must determine optimal conditions for each antigen of interest. Many protocols for staining individual antigens, as well as useful tips and troubleshooting guides for immunohistochemistry, can be found at the IHC World web site (<a href="http://www.ihcworld.com/" target="_blank">http://www.ihcworld.com/</a>).</p>

[STEPS]
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TITLE: SHORT: a human brain tissue clearing and labeling protocol for 3D recostruction with LSFM
AUTHORS: Luca Pesce, Marina Scardigli, Vladislav Gavryusev, Annunziatina Laurino, Giacomo Mazzamuto, Giuseppe Sancataldo, Ludovico Silvestri, Christophe Destrieux, Patrick R. Hof, Irene  Costantini, Francesco S. Pavone

[DESCRIPTION]
This protocol is a detailed version of the SHORT clearing and labeling method optimized by the European Laboratory for Non-Linear Spectroscopy - University of Florence  -Italy, which describes the process to optically clear and fluorescently label human brain volumetric slabs, permitting the 3D reconstruction of the tissue at micrometer-resolution using light-sheet fluorescence microscopy (LSFM).

Note: Research reported in this publication was supported by the European Union's Horizon 2020 research and innovation Framework Programme under grant agreement No. 654148 (Laserlab-Europe); European Union's Horizon 2020 Framework Programme for Research and Innovation under the Specific Grant Agreement No. 785907 (Human Brain Project SGA2) and No. 945539 (Human Brain Project SGA3); Italian Ministry for Education in the framework of Euro-Bioimaging Italian Node (ESFRI research infrastructure); "Fondazione CR Firenze" (private foundation) and the BRAIN Initiative Cell Census Network of the National Institutes of Health under award number U01 MH117023. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

[STEPS]
1.
TITLE: ConA Cover Slide Preparation
AUTHORS: Mathias Hammer, Ammeret Rossouw, Azra Lari, Ben Montpetit, David Grunwald

[DESCRIPTION]
Concanavalin A is used to attach yeast cell to surfaces. This protocol describes the preparation of cover slides for high resolution fluorescence microscopy.

[BEFORE_START]
Have the following solutions premixed:

Concanavalin A (ConA) solution:
Concentration: 1 g/l
resolve 10 mg ConA in 10 ml distilled water, vertex
store at -20 °C 


[STEPS]
SECTION: Cover slide preconditioning
1. Rinse a 25mm cover slide No. 1.5 or No. 1.5H. 
Dry with Kim wipe, remove leftover driplet with optical tissue.

SECTION: Cover slide preconditioning
1.1. Plasma clean the cover slide to a hydrophilic negative charged surface.
Quorum Emitech K100X glow discharger at 25 mA 45 s 
SECTION: Cover slide preconditioning
1.2. Place the cover slide in the cell chamber.
SECTION: ConA slide preparation
2. Defrost the stock ConA of a concentration of 1 mg/mL 

SECTION: ConA slide preparation
2.1. Pipette 1 mL ConA solution into the cell chamber.

SECTION: ConA slide preparation
2.2. Let the ConA settle, covered with Kim wipe for 10 min at the bench.

SECTION: ConA slide preparation
2.3. Remove the ConA from the chamber back in its container.
Let the cover slip dry, covered with Kim wipe for 20 min .
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>A protocol for yeast colony PCR, starting with intact cells. Can be used to analyze genomic and plasmid DNA, with PCR products up to 2 kb being no problem. It doesn't get easier than this: pick a small amount of cells from a plate into water, lyse cells at 99C for 5 min. Use the lysed cells as template by adding to 1/10th final volume in a PCR. We use this protocol for diagnostics (Phire HS II enzyme) and for Sanger sequencing (Phusion HS II or NEB Q5 enzyme). The main reasons why others might not get this protocol to work could be due to using too many cells and/or not using pure water for the cell lysis.  Good luck, and have fun! </p>
<p> </p>
<p>See the 'BEFORE START' section of this protocol for photographs that show how to pick cells from a colony and make a suspension.  See the 'WARNINGS' section for important points and trouble shooting. </p>

[BEFORE_START]
<p>Below are a few photographs of picking cells from a colony and making cell suspensions. Illustrated in the last photo are three cell suspension densities, one of them being just right. We believe a problem of our protocol failing for some researchers is using too many cells, perhaps with the idea of  'the more the better.'  If you are having problems, try using less cells.</p>
<p> </p>
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/h3ifqvw.jpg" data-src="https://s3.amazonaws.com/pr-journal/h3ifqvw.jpg" data-ofn="Colony PCR pasteup.jpg" /></p>

[STEPS]
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TITLE: SARS-CoV-2 Genomic Variation - African perspective
AUTHORS: Olabode Omotoso

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the methodology for the acquisition and analysis of SARS-CoV-2 genomic sequences.</div></div>

[STEPS]
?. [Data Acquisition]
Mine and analyze SARS-CoV-2 genomic sequences from the Global Initiative on Sharing All Influenza Data (GISAID) database (epicov.org). Use sequences filtered as “high coverage only, Homo sapiens, complete, all clades and low coverage excl”, with patient’s status, "Africa".
?. [Data Acquisition]
Obtain the patient’s age of all the sequences to determine the age distribution of the infected patients.
?. [Data Acquisition]
Obtain country data of the number of confirmed cases, recoveries, reported deaths due to COVID-19 from Worldometer (worldometers.info) and WHO database (covid19.who.int).
?. [Data Acquisition]
Obtain the number of tests done per country and each nation’s population from the Worldometer database.
?. [Data Acquisition]
Obtain the age distribution of countries with the highest prevalence of COVID-19 cases from the World Factbook (www.cia.gov).
?. [Sequence and Mutational Analysis]
Use the mined SARS-CoV-2 viral sequences to analyze the genomic variability since the index case of the COVID-19 pandemic in Africa in February 2020 to identify the frequency and spread of mutations in the African population.
?. [Sequence and Mutational Analysis]
Assess and evaluate the evolution of the COVID-19 outbreak with respect to the transmission in the mutational hotspots on the GISAID web interface (www.epicov.org). Focus on recurrent mutations observed as they are likely to confer viral-host structure-function relationship promoting higher transmission rate.
?. [Determination of Testing, Fatality and Recovery Rate]
Determine the testing rate for each African country as a percentage of the total test done from the country’s population.
Testing Rate (%) = (Total COVID-19 tests done/Country's population) x 100
?. [Determination of Testing, Fatality and Recovery Rate]
Determine the fatality rate as a percentage of total reported deaths due to COVID-19 from each country’s number of confirmed cases.
Fatality Rate (%) =(Total number of COVID-19 deaths reported/Number of confirmed cases) x 100
?. [Determination of Testing, Fatality and Recovery Rate]
Determine the recovery rate as a percentage of the number of infectious patients who recovered from all reported confirmed cases in each country.
Recovery Rate (%) = (Total number of COVID-19 infected patients who recovered/Number of confirmed cases) x 100
TITLE: RT-LAMP Reaction
AUTHORS: Noah Toppings

[STEPS]
?. Add 9.5 µL extracted RNA + 15 µL resuspension buffer + 0.5 µL of dye to a lyophilized reaction pellet.
?. Mix reaction by pipetting.
?. Add 30 µL mineral oil on top of reaction.
?. Spin down the strip of tubes.
?. Incubate at 61 ºC for 45 minutes.
?. Visualize reactions under a transilluminator.
TITLE: CLINICAL CHARACTERISTICS, PATTERNS OF USE, AND INCIDENCE OF ADVERSE EVENTS IN PATIENTS WITH NONVALVULAR ATRIAL FIBRILLATION TREATED WITH ORAL ANTICOAGULANTS
AUTHORS: Jorge        Machado Alba

[DESCRIPTION]
Purpose: The aim was to analyze the characteristics, treatment patterns, persistence, and clinical outcomes of Colombian patients with Non-Valvular Atrial Fibrillation (NVAF) under treatment with oral anticoagulants (OAs).
Patients and methods: Retrospective cohort in patients with NVAF, aged ≥18 years and of any sex, the first prescription of an OA (index) between January/2013 and June/2018, with a follow-up until June/2019. Data from the clinical history, patient characteristics, pharmacological variables, treatment switch, cerebrovascular events and safety were searched. Descriptive analyzes were carried out.
Results: A total of 2076 patients with NVAF were included. The 57.0% of patients were women and the mean age was 73.3±10.4 years. Patients were followed for a mean of 2.3±1.6 years. 8.7% received warfarin before the index date. The most frequent OA was rivaroxaban (n=950; 45.8%), followed by warfarin (n=459; 22.1%) and apixaban (n=405; 19.5%). Hypertension was present in 87.5%, diabetes mellitus in 22.6% and severe renal failure in 5.2%. The mean CHA₂DS₂-VASc Score was 3.6±1.5. The main efficacy and safety outcomes were stroke (3.1%) and gastrointestinal bleeding (2.0%) respectively. From these, most events were in patients using warfarin at index (3.5% for stroke and 4.1% for gastrointestinal bleeding). The 18.0% of patients switched index OA during follow-up, while 11.7% discontinued the treatment. 
Conclusion: The patients with NVAF in this study were mainly older adults with multiple comorbidities and high CHA₂DS₂-VASc score. The frequency of clinical events was similar to other observational studies, with low percentage of discontinuation.

[STEPS]
TITLE: Activation of Simvastatin
AUTHORS: Dennis Juarez, dfruman

[DESCRIPTION]
Activation of simvastatin lactone to active simvastatin acid by hydrolysis of lactone ring with NaOH.

[STEPS]
SECTION: Preparation
1. For every 8.4 mg of simvastatin sodium salt, dissolve in 0.2 mL of 100% ethanol.
SECTION: Preparation
2. Add 30 uL of 1N NaOH for every 8.4 mg of simvastatin sodium salt.
SECTION: Preparation
3. Heat the solution for at 50 °C for 2 hours.

SECTION: Preparation
4. Neutralize the solution to pH 7.2 with 1N HCl.
SECTION: Preparation
5. Bring to a volume of 1 mL per 8.4 mg of simvastatin dissolved in Step 1 with distilled H2O.
SECTION: Preparation
6. Dilute 1:1 with DMSO to yield a 10mM solution with 50% DMSO, and 10% EtOH in H2O.
SECTION: Preparation
7. Store at -80 °C
TITLE: Caspase activity assays
AUTHORS: Kiichi Hirota, Yoshiyuki Matsuo

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Cells were seeded into 96-well plates and incubated overnight.
?. The following day, cells were treated with reagents for varying lengths of time.
?. 100 μl of Apo-ONE® Caspase-3/7 Reagent was added to each well.
?. Cells were incubated at room temperature for 1 h.
?. The luminescence of each well was measured using an EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA).
?. Caspase activity was then calculated by comparing the levels of luminescence of the treated cells with those of the control cells (incubated without drugs), with the latter defined as 100%.Assays were performed in triplicate at least twice. Data were expressed as mean ± standard deviation (SD).