eamag/protocols-with-steps huggingface dataset:
TITLE: protocol Animal-Assisted Therapy
AUTHORS: Mohammad  Tahan

[DESCRIPTION]
Human-animal interactions are considered as being valuable and beneficial for the psychological health. Recently Animal-Assisted Therapy (AAT) has been included for client-therapist interaction.

[STEPS]
SECTION: Animal-Assisted Therapy
1. Session one	Introduction, briefing, primary assessing and introduction to the course
SECTION: Animal-Assisted Therapy
2. Session two	Giving an introduction about animals and their specific characteristics
SECTION: Animal-Assisted Therapy
3. Session three	Taking children to the place that animals were kept
SECTION: Animal-Assisted Therapy
4. Session four	Spending time in the place that animals were kept, talking about them and how to communicate with them
SECTION: Animal-Assisted Therapy
5. Session five	Convincing children to come closer and touch and pet them
SECTION: Animal-Assisted Therapy
6. Session six	Playing with animal and feeding them
SECTION: Animal-Assisted Therapy
7. Session seven	Participation in activities like washing and feeding the animals
SECTION: Animal-Assisted Therapy
8. Session eight	Summarizing, giving feedback, and post-test
TITLE: Wire Hang Assessment
AUTHORS: Tim Sampson, Ian N Krout, Alexandria White

[DESCRIPTION]
The wire hang test for mice is a simple and cheap way to asse motor and neuromotor pathologies in the mouse model. Described herein is an adapted protocol from Deacon 2013.

[STEPS]
SECTION: Apparatus
1. A wire hang apparatus can be made easily for use in this protocol and requires very little material (see Figure 1 for example). The apparatus should be easily traversed by the mice, meaning the spacing of the bars should be close enough to allow for free and easy movement of the mice and the width of the bars should be thin enough to allow for full grip by the paws. According to a protocol by Deacon, 2013 the ideal measurements are as follows. “The inverted screen is a 43 cm square of wire mesh consisting of 12 mm squares of 1 mm diameter wire (Figure 1). It is surrounded by a 4 cm deep wooden beading (which prevents the occasional mouse which attempts to from climbing on to the other side)”
 

SECTION: Acclimation
3. Bring mice up from vivarium in their home cage to acclimate for 1h in the behavior room (or whatever location assessment takes place) prior to wire hang assessment. 
SECTION: Assessment
4. Randomize the order of mice for assessment and record the coding in the lab notebook.
SECTION: Apparatus
2. This apparatus should be suspended above a cage filled with bedding to provide the mice with a safe place to land when descending from the wire hang (Figure 2.)

 



SECTION: Assessment
5. Begin by taking the first mouse out of its home cage and place the mouse, stomach down, onto
the top of the wire hang apparatus. Make sure to the place the mouse facing away
from you in the center of the wire hang screen. 
SECTION: Assessment
6. Slowly rotate the wire hang apparatus with the head of the mouse declining first (away from
your body) making a full 180 degree rotation and placing the apparatus back in the start
position (the mouse should now be hanging with its back facing the bench)
(Figure 3).

 


SECTION: Assessment
7. Start a stopwatch and record the amount of time the mouse is able to hang on the
screen. If the mouse reaches 120 seconds remove it from the screen and record
this time. 
SECTION: Assessment
7.1. Note that mice may try to climb up and over the apparatus, if this happens, reposition the mouse and flip the screen again.
SECTION: Assessment
7.2. The mice may also try to grab onto the side of the cages used to suspend the screen
causing early falling, make note of this.
SECTION: Assessment
8. Repeat this 2x for a total of 3 trials, allowing the mouse to recover by roaming the
cage with bedding for ~1 minute between trials. 
SECTION: Assessment
9. Move onto the next mouse until all mice are assessed.
SECTION: Assessment
10. Scores are recorded as either the average of 3 trials or as independent trials showing
all 3 runs. In a normal situation – the mice will be able to hang for less and
less time each subsequent trial.
TITLE: NIBS using tDCS to mitigate aggression 
AUTHORS: Paola Di Maio

[DESCRIPTION]
Protocol Description:  Non Invasive Neuromodulation procedure using tDCS (transcranial direct current stimulation) to mitigate aggressive behavior in healthy adults
Condition: tendency to exhibit overly aggressive behavior
Safety: The procedure must be administered by trained personnel, and the subject must obtained medical clearance
Contraindications: Do not administer this procedure if the subject has metallic implants near the
electrode sites; is epileptic; has unstable medical conditions; neurological, cardiovascu-
lar, or psychiatric illness; participates in another noninvasive brain
stimulation study on the same day; has history of adverse reactions to tDCS;
Do not apply electrodes to skin with lesions


tDCS intervention. using a battery-driven, constant-current stimulator .

DLPFC  pre frontal cortex
Tdcs  transcranial direct current stimulation

[BEFORE_START]
Two hours before or after main meals
wearing no metal objects
comfortable clothing
sitting in semi reclined position

[GUIDELINES]
Safety Warning: The procedure Must be administered by trained personnel ; the subject must  obtain medical clearance

[STEPS]
SECTION: Safety Warnings
6. Use tDCS equipment and accessories compliant with international standards, by trained personnel
7. In pre clinical studies, the procedure can mitigate Aggressive behaviour
SECTION: Protocol Description
1. Two anodal electrodes are placed over the DLPFC bilaterally (F3 and F4) according to the International 10 –20 EEG system.
SECTION: Protocol Description
2. A constant current of 2 mA (1 mA to each DLPFC site) is applied for 20 min through saline-soaked sponge electrodes (5  5 cm).
SECTION: Protocol Description
3. A single extracephalic cathodal electrode (5  7 cm) is placed at the posterior base of the neck to minimize unintentional effects of inhibitory stimulation on brain Activity.
SECTION: Protocol Description
4. Following standard tDCS protocol, stimulation commences after 30 s ramp-up period. The current is ramped down over the last 2 s.
SECTION: Protocol Description
5. Check the subject throughout the procedure and discontinue if the subject shows discomfort
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This year´s interlab study allows participants to fulfil Bronze medal requirements. </p>
<p>For this purposes a set of experiments has to be performed, which in the end will be compared and validated with other team´s data. </p>
<p>Part of this Challenge are the Calibration protcols. </p>
<p> </p>

[BEFORE_START]
<p>Make sure to adjust the temperature of your plate reader to room temperature (22°C-25°C) before measurement.</p>

[GUIDELINES]
<p>Make sure to always use the same plates, volumes and settings for the measurement as will be used for the other calibrations and the cell measurements. </p>
<p>Also take care to remain constant with your pipetting techniques. </p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The Seattle Children's Hospital launched a pioneering clinical trial of <a href="https://www.creative-biolabs.com/car-t/cellrapeutics-chimeric-antigen-receptor-car-technology.htm" target="_blank" rel="noopener noreferrer">chimeric antigen receptors</a> (CAR) T-cell immunotherapy for relapsed or refractory HER2-positive CNS tumors in children and young adults. Among them, CAR-T cells will be delivered directly to the brain. In a Phase 1 trial of BrainChild-01, depending on the location of the tumor, CAR-T cells will be catheterized into the cavity or CNS ventricular system where the tumor has been removed.</p>

[STEPS]
?.
TITLE: UH-PAM Protocol
AUTHORS: Seonggun Joe, Massimo Totaro, Hongbo Wang, Lucia Beccai

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol outlines the fabrication process of UH-PAM, and it prompts you with the details to repeat and reproduce our works.</div></div>

[STEPS]
?. Please download and unzip "UH-PAM Protocol.zip" in the attached file.
?. Cut a commercial polyurethane open-cell foam and Plexigals as attached files (UH-PAM_2D Layers.dwg)We recommend 2D laser cutter (Versa LASER VLS 3.5), in order to properly cut them.For the print parameters, please follow the table, as below:  ABCDE1POWERSPEEDPPIZ-axis2Segment foam60%30%1,00012.6mm3Bridge foam60%30%1,0006.4mm4Flexiglas rings100%10%1,0001mm
ABCDE1POWERSPEEDPPIZ-axis2Segment foam60%30%1,00012.6mm3Bridge foam60%30%1,0006.4mm4Flexiglas rings100%10%1,0001mm
?. Print the molds (in the attached files, Mold(1) to (4).stl) by using a 3D printer.
?. Pour the mixed liquid into mold (1) and (3), and assemble it with mold (2) and (4), respectively.
?. After curing for 15 hours, detach the skins from the molds
?. Bond the segment foam with the bridge foam, and build a foam structure. Here, do not pour the silicone and use a silicone adhesive material (SIL-Poxy® adhesive, Smooth-On Inc).Otherwise, the foam absorbs the silicone, and its mechanical property would be changed.
?. Bond all skin parts and the foam structure, by brushing the Dragonskin 30.
?. After curing the silicone, bond both halves of the skin.
TITLE: BICAN workflow protocol
AUTHORS: Satrajit Ghosh

[DESCRIPTION]
A test protocol.

[STEPS]
SECTION: Neuropixels workflow
1. Surgery  

SECTION: Neuropixels workflow
1.1. Mouse head-fixed and anesthetized
SECTION: Neuropixels workflow
1.2. Headframe implant
SECTION: Neuropixels workflow
1.3. Craniotomy and durotomy
SECTION: Neuropixels workflow
1.4. Cranial window implant
SECTION: Neuropixels workflow
1.5. Photo documentation
SECTION: Neuropixels workflow
1.6. Health and window check
SECTION: Neuropixels workflow
2. Intrinsic imaging
SECTION: Neuropixels workflow
2.1. Mouse head-fixed and anesthetized
SECTION: Neuropixels workflow
2.2. Vascular imaging
SECTION: Neuropixels workflow
2.3. Visual area mapping
SECTION: Neuropixels workflow
2.4. Auto segmentation / Auto annotation
SECTION: Neuropixels workflow
2.5. QC of ISI imaging data
SECTION: Neuropixels workflow
3. Habituation
SECTION: Neuropixels workflow
3.1. Handling and awake head fixation
SECTION: Neuropixels workflow
3.2. Passive viewing of stimuli
SECTION: Neuropixels workflow
3.3. On experimental rig
TITLE: Variant functionalization by localization
AUTHORS: jessechao 

[STEPS]
?. [Cell culture and transfection]
Grow cells  to sub confluent.For example, using MCF10A cells, seed cells at 2x105 cells per well in a 6-well plate 24h before transfection and incubate overnight.
?. [Cell culture and transfection]
Transfect cells according to manufacturer's recommendations. For example, we used lipofectamine 2000.
?. [Cell culture and transfection]
Clean, sterilize and dry coverslips. You may need to treat the coverslips with poly-L-lysine to encourage cell adhesion. Place the coverslips in the appropriate plasticware.For example, we used 22x22mm glass coverslips (Fisher) in 6-well plates (Corning).
?. [Cell culture and transfection]
The next day, gently wash the coverslips with PBS. Verify transfection efficiency by checking under a fluorescent microscope. If the transfection efficiency is satisfactory (i.e. > 10%), continue.
?. [Immunofluorescence]
This section shows the steps of a typical immunofluorescence experiment. It generally involves fix -> permeabilize -> block -> primary antibody -> secondary antibody ->mount. All steps are at room temperature unless stated otherwise.
?. [Immunofluorescence]
Aspirate the PFA and wash 3x with PBS. Permeabilize cells by adding 0.1% Triton X-100 (in PBS) and incubate for 5 min.
0 Room temperature
?. [Immunofluorescence]
Aspirate the Triton X-100 and wash 3x with PBS. Block by using the appropriate blocking solution e.g. 10% goat serum or BSA (in PBS). Incubate for 10 min.
0 Room temperature
?. [Immunofluorescence]
Wash the cells 3x with PBS. Fix cells by adding 4% PFA (in PBS) and incubate for 10 min.
0 Room temperature
?. [Immunofluorescence]
Aspirate the blocking solution and wash 3x with PBS. Add primary antibody (in blocking solution). In our experiments, we used rabbit anti-PTEN antibody (138G6, Cell Signaling Technology) at 1:100 in blocking solution. Incu bate overnight.
4 °C
?. [Immunofluorescence]
Wash the coverslips 3x with PBS. Add secondary antibody (in blocking solution). In our experiments, we used mouse anti-rabbit Alexa Fluor 568-conjugated antibody (Invitrogen) at 1:100 in blocking solution. Incubate for 1 hr.
0 Room temperature
?. [Immunofluorescence]
Wash the coverslips 3x with PBS. Incubate the coverslips with DAPI (in PBS) if you want to stain the nuclei for 5 min then wash with PBS.
?. [Immunofluorescence]
Mount the coverslips onto glass microscopy slides by using your preferred mounting media. We used ProLong Gold antifade mountant (Thermo Fisher Scientific).
?. [Microscopy]
Acquire images with your favorite microscopy setup. An automated microscope is not required to proceed to image analysis using our MAPS software, but will certainly accelerate image acquisition. In our experiments, we used a Cellomics Arrayscan (Cellomics Inc.). using a 20x objective. 500 images were acquired per coverslip at 3 channels (green/ red/ blue) per image.
?. [Immunofluorescence]
After waiting ~30min or more, seal the slides with clear nail polish.
TITLE: Characterization of iPSC 
AUTHORS: Celeste Karch, Rita Martinez, Jacob Marsh

[STEPS]
?. Coat T25 flask, chamber slide and plates with Matrigel  prior to passaging.
?. Aspirate media from cell culture.
?. Wash with  PBS per well and aspirate.
1 ml
?. Add Accutase per well of 6 well plate.
1 ml
?. Incubate infor 3-4 minutes.
?. Collect cells from 2 wells with  DMEM/F12 per well and transfer into 15mL conical A.
3 ml
?. Collect cells from 4 wells with  DMEM/F12 per well and transfer into 15mL conical B.
3 ml
?. Spin cells at 750 rpm for .
?. Aspirate media from each tube.
?. [Tube A]
To 15mL conical tube A, add  mTesR1 and distribute cells.
2 ml
?. [Tube A]
Karyotype: Addof mTesR1 to T25 flask, then add cells .
2 ml
500 µl
?. [Tube A]
gDNA pellet: cells in 1.7 mL tube, spin down at max speed for , aspirate media, store in .
500 µl
?. [Tube A]
RNA pellet: cells in 1.7 mL tube, spin down at max speed for , aspirate media, store in
900 µl
?. [Tube A]
ICC: Dilute  cells in  mTesR1. Plate cells per well in 4 wells of a chamber slide.
100 µl
750 µl
200 µl
?. [Tube B]
To 15mL conical tube B, add mTesR1 and of 2x Freezing Media (20% DMSO, FBS).
4 ml
4 ml
?. [Tube B]
Add cell suspension to each vial (1 well will freeze down into approximately 2 vials).
1 ml
Cryovials need to be labeled with the following before freezing down: Cell Type Line Name Passage # Date Your Name
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><span style="font-weight: 400;">Esta prueba evalúa la actividad hemolítica de péptidos o moleculas sobre eritrocitos humanos como criterio de selectividad ante las células eucariotas. </span></p>
<p> </p>
<p><span style="font-weight: 400;">La realizacion de este protocolo fue posible gracias al apoyo del departamento administrativo de ciencia tecnología e innovacion, Colciencias a traves del proyecto 111356933173 convocatoria569-2012. </span></p>

[BEFORE_START]
<p><strong><em>Preparar solución de lavado: </em></strong></p>
<p> </p>
<p><span style="font-weight: 400;">Buffer PBS 1X: 130 mM NaCl, 3 mM KCl, 8 mM Na2HPO4 y 1,5mM KH2PO4 pH 7,4.</span></p>
<p> </p>
<p><span style="font-weight: 400;">Se puede utilizar como solución alternativa: 150mMKCl, 5mM Tris–HCl, pH 7.4</span></p>
<p> </p>
<p><strong><em>Solución de control hemolisis: </em></strong><span style="font-weight: 400;">Tritón X100 solución al 1% o al 0,1%. </span></p>
<p> </p>
<p><strong><em>Blanco del ensayo: </em></strong>Utilizar <span style="font-weight: 400;">solución de lavado o diluyente de la muestra. </span></p>
<p> </p>
<p><strong><em>Preparar las muestras a evaluar.</em></strong></p>

[GUIDELINES]
<p>Este procedimiento consta de la extracción de la sangre, lavado y dilución de eritrocitos y finalmente de la preparación de la muestra que se va a poner en contacto con los eritrocitos para evaluar su actividad hemolítica.</p>
<p>El tiempo estimado es de 4 a 5 horas contabilizando desde el momento en el que se extraen los eritrocitos hasta el momento en el que se lee la placa en el espectrofotometro por turbidez.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Parse Evercode WT -- University of Minnesota TMCs
AUTHORS: Laura J Niedernhofer, David A Bernlohr

[DESCRIPTION]
Combinatorial barcoding is an instrument-free method used to generate single-cell and single-nucleus RNA-sequencing libraries. Outlined here are the methods used to isolate single-nuclei from frozen tissue; to fix nuclei following isolation; to ligate nuclei-specific barcodes to transcript cDNA by iteratively splitting and pooling nuclei suspensions between multiple 96-well plates via the Evercode WT system; and to prepare barcoded cDNA for sequencing by synthesis (SBS). The following protocol has been adapted from protocols developed by 10x Genomics, Parse Biosciences, and Illumina to be used at the University of Minnesota TMCs in collaboration with the University of Minnesota Genomics Center. These protocols are owned by their respective companies and are subject to periodic revision.


[STEPS]
SECTION: Single Nuclei Dissociation
1.   

SECTION: Nuclei Fixation Protocol
2.   

SECTION: Evercode WT Protocol
3.   
 


 

SECTION: FASTQ Generation
4. BCL data from Illumina sequencer is demultiplexed and converted into FASTQ format using bcl2fastq version 2.20.0
TITLE: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
AUTHORS: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
We adapted a previously-described method (Pantazis et al., 2022) for employing Piggybac transfection to stably express doxycycline-inducible NGN2 in human iPSCs. After stable integration of NGN2, proceed to differentiate iPSCs using protocol “iNeuron differentiation from human iPSCs.” 

[GUIDELINES]
Citations:

Pantazis, C.B., Yang, A., Lara, E., McDonough, J.A., Blauwendraat, C., Peng, L., Oguro, H., Zou, J., Sebesta, D., Pratt, G., et al. (2022). A reference induced pluripotent stem cell line for large-scale collaborative studies. BioRxiv 2021.12.15.472643.

[STEPS]
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
1. Culture iPSCs in a 10 cm dish coated with Growth Factor Reduced Matrigel (Corning) and feed daily with Essential 8 media (ThermoFisher). 
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
2. Passage iPSCs with warm Accutase into Essential 8 media with 10 micromolar (µM) ROCK inhibitor. Plate 800,000 iPSCs into one Matrigel-coated well of a 6-well plate.
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
3. 3 - 6 hours after plating, cells should be healthy and attached. Perform transfection using Lipofectamine Stem and a 2:1 ratio of donor plasmid to transposase:

 

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
4. Check for transfection efficiency (BFP-labeled cells) on the next day using fluorescence microscopy.
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
5. 4320 min after transfection, select for transfected iPSCs with 0.5 μg/ml puromycin.

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
6. Cryopreserve selected iPSCs with 

 

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
4.1. Passage iPSCs with Accutase to a 10 cm dish when cells are confluent enough for splitting. 
 

SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
5.1. Confirm purity of surviving transfected cells with fluorescence microscopy. When population is pure, withdraw puromycin. 
SECTION: Piggybac-mediated stable expression of NGN2 in iPSCs for differentiation into excitatory glutamatergic neurons
6.1. Proceed to culture and induction to neuronal fate using doxycycline (see “Protocol: iNeuron differentiation from human iPSCs”).
TITLE: VU Biomolecular Multimodal Imaging Center (BIOMIC) kidney characterization pipeline for tissues collected through the Cooperative Human Tissue Network (CHTN)
AUTHORS: Elizabeth Neumann, Jamie Allen, Maya Brewer, David Anderson, Mark De Caestecker, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We aim to develop high resolution, chemically informative imaginig methodologies for building an atlas of human organs, such as the kidney.</div><div class = "text-block">Scope:</div><div class = "text-block">Provide an overview of the methods used by the Vanderbilt Tissue Mapping Center as part of the Human Biomolecular Atlas Program (HuBMAP, NIH Common Fund) and contextualize individual protocols within our larger workflow.</div></div>

[STEPS]
?. Collection of post-surgical tissue.Collection:  dx.doi.org/10.17504/protocols.io.7gehjte
?. Stabilize and freeze tissues.Freezing Tissue:  dx.doi.org/10.17504/protocols.io.6wghfbw
?. Intial Rapid Pathology Assessment of Kidney TissueStaining: dx.doi.org/10.17504/protocols.io.4qngvveAssessment: dx.doi.org/10.17504/protocols.io.9dph25n
?. Cryosection tissues into micrometer thick sections, alternating between thaw mounting onto indium tin-oxide and positively charged glass slides (proceed to step 4), or collecting several tissue sections within an microcentrifuge tube for proteomics analysis.Cryosectioning:  dx.doi.org/10.17504/protocols.io.7ethjen
?. Perform autofluorescence microscopy on all tissue sections before IMS (step 5) or MxIF analysis (step 9)Autofluorescence:  dx.doi.org/10.17504/protocols.io.7e3hjgn
?. Coat tissue sections with MALDI matrix for IMS analysis.Matrix Application:  dx.doi.org/10.17504/protocols.io.4srgwd6
?. Perform high resolution IMS analysis of matrix coated tissue sections.IMS:  dx.doi.org/10.17504/protocols.io.7gdhjs6
?. Perform fluorescence microscopy to visualize laser ablation spots.Post IMS AF: dx.doi.org/10.17504/protocols.io.879hzr6
?. Annotation of Lipids from IMS DataCal & Annotate: dx.doi.org/10.17504/protocols.io.864hzgw
?. Remove MALDI matrix and perform PAS staining.PAS Staining:  dx.doi.org/10.17504/protocols.io.4qngvve
?. Alternatively, MxIF can be peformed after step 4.Antibody labeling:  dx.doi.org/10.17504/protocols.io.667hhhnMxIF:  dx.doi.org/10.17504/protocols.io.665hhg6
?. Registration of autofluorescence images from both IMS and MxIF sections allow for the direct correlation of the two orthogonal approaches.Registration: https://dx.doi.org/10.17504/protocols.io.bed2ja8ePublication:  https://doi.org/10.1021/acs.analchem.8b02884
?. MALDI imaging mass spectrometry data processing.Data Processing: https://dx.doi.org/10.17504/protocols.io.bed3ja8n
?. RNA Assessment from tissue.RNA extraction: dx.doi.org/10.17504/protocols.io.86nhzde
?. Perform LC based Proteomics on adjacent tissue sections and/or continue to Step 6.Sample Preparation:   dx.doi.org/10.17504/protocols.io.67nhhmeData Acquisition: dx.doi.org/10.17504/protocols.io.bfsjjncnData Analysis:  dx.doi.org/10.17504/protocols.io.bfshjnb6