eamag/protocols-with-steps huggingface dataset:
TITLE: Staining/Dehydration 10 micron Rat Heart 
AUTHORS: Shaina Robbins, Sean Nieves

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

[STEPS]
?. [Slide retrieval]
Remove slide from  freezer. Keep slide covered and allow to come to room temperature for  or until frost on slide dissipates.
-80 °C
?. [Fixation]
Submerge slide into cold acetone (kept in  freezer) for
-20 °C
?. [OCT removal]
Gently dip slide 10 times into 1x PBS or until OCT appears to be washed off
?. [Staining]
Submerge slide into 0.1% Cresyl Violet for
?. [Rinse]
Gently rinse slide by dipping into H2O 10 times
?. [Rinse]
Gently dip slide 10 times into 1x PBS
?. [Dehydration ]
Dip and agitate slide 2 times into 95% EtOH
?. [Dehydration ]
Dip and agitate slide 2 times into 100% EtOH
?. [Dehydration ]
Submerge slide into 100% EtOH for
?. [Slide retrieval]
Gently dip slide 2 times into Xylene
Flammable liquids (Category 3), H226 Acute toxicity, Inhalation (Category 4), H332 Skin irritation (Category 2), H315 Eye irritation (Category 2A), H319 Carcinogenicity (Category 2), H351
?. Submerge slide into Xylene for
Flammable liquids (Category 3), H226 Acute toxicity, Inhalation (Category 4), H332 Skin irritation (Category 2), H315 Eye irritation (Category 2A), H319 Carcinogenicity (Category 2), H351
?. Allow slide to dry under chemical fume hood for Proceed to LCM/LMD
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The FlowCam can be used to take quick cell density measurements of liquid samples. </p>

[BEFORE_START]
<p>Before you begin, make sure that</p>
<ol>
<li>the appropiate flow cell and syringe are mounted</li>
<li>appropiate objective and columnator, if needed, are in place</li>
</ol>
<p>The above two usually default to the following configuration:</p>
<table style="height: 58px;" width="266">
<tbody>
<tr>
<td style="width: 125px;">Flow Cell</td>
<td style="width: 125px;">
<p>100 um (fc100)</p>
</td>
</tr>
<tr>
<td style="width: 125px;">Syringe</td>
<td style="width: 125px;">0.5 mL</td>
</tr>
<tr>
<td style="width: 125px;">Objective</td>
<td style="width: 125px;">10x with columnator</td>
</tr>
</tbody>
</table>

[STEPS]
?. 
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TITLE: Preparation of Gelatin Slide Coating Solution
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the preparation of gelatin solution to be used for coating glass microscope slides.</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: Pole Test to assess motor coordination  in parkinsonian mice
AUTHORS: natalia.lopezgonzalezdelrey, Zachary Gaertner

[DESCRIPTION]
The pole test evaluates the motor coordination ability of a mouse by having them grasp a pole and then descend to its home cage.



[STEPS]
SECTION: Pole Test
1. Total duration: 1 day
Three consecutive trials per animals are run.
SECTION: Protocol
2. Place the animal cage in the testing room to allow the animals to acclimate. 60 min 
SECTION: Protocol
4. Return the mouse to its cage.
SECTION: Analysis
5. The time required for the animals to orient themselves facing in a downward direction (time to T turn) and to descend to the base of the pole (total time) is recorded.

Mean times per mouse are compared across groups. 


SECTION: Protocol
3. Mice are placed with their head oriented upward at the top of a 16in tall, 1⁄2in diameter wooden pole coated in tape to allow gripping. The animals normally orient themselves downward and then descend the length of the pole in order to return to their home cage.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Female larvae of the Aedes aegypti mosquito outcompete male larvae for food.  Male larvae pupate earlier and at smaller masses in vials where competition is most intense.  Competition at the higher densities causes the food/larva to appear lower than the equivalent mg/larva at lower densities for both sexes.  The total food per vial appears to change the nature of the exploitative competition among females, which affects the competition among males.  No interference competition was observed; females may dominate the competition by their larger size (increased filtering ability) and by retaining food particles within their guts to extract a larger fraction of their nutrients.  Pupal mass for both sexes is primarily affected by food/larva; density affects the pupal mass through competition and the total food/vial (the interaction between food and density) and this interaction is different for the two sexes.  Age at pupation is more affected by density than by food/larva and the interaction between food and density also differs across the two sexes for this variable.  The nature of competition between and within the sexes at various food levels and densities is described.  The implications of this complex response to food level and density among larvae on the ecology of this species is considered. </p>

[GUIDELINES]
<p>It is easier to create a standardized solution of baker's yeast and dilute it down to the concentration necessary to add the calculated total mg per vial for each treatment.  However, the yeast must be completely dissolved in the solution and it must be repeatedly stirred to keep it from clumping or settling.</p>

[STEPS]
?. 
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TITLE: UC Davis - Non-Esterified Fatty Acids Protocol
AUTHORS: Peter Havel

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
The Wako enzymatic method relies upon the acylation of coenzyme A (CoA) by the fatty acids in the presence of added acyl-CoA synthetase (ACS). The acyl-CoA thus produced is oxidized by added acyl- CoA oxidase (ACOD) with generation of hydrogen peroxide, in the presence of peroxidase (POD) permits the oxidative condensation of 3-methy-N-ethyl-N(β-hydroxyethyl)-aniline (MEFA) with 4-aminoantipyrine to form a purple colored adduct which can be measured colorimetrically at 550 nm.</div></div>

[STEPS]
?. Reconstitute Color Reagent A with 50 ml of Solvent A and Color Reagent B with Solvent B.
?. Add 5 μl of calibrator and sample to each well.
?. Add 200 μl of Reagent A to each well. Incubate at 37°C for 5 minutes. Read at 560 nm.IMPORTANT: Make sure not to add any bubbles to the wells when dispensing reagents, this will interfere with reading in the platereader.
?. Add 100 μl of Reagent B to each well. Incubate at 37°C for 5 minutes. Read at 560 nm.
?. Subtract blank readings from final readings. The assay will be linear so the unknown samples can be calculated as (Sample Absorbance ÷ Calibrator Absorbance) × Calibrator Concentration.
TITLE: HuBMAP | GE/Vanderbilt MALDI IMS and Cell DIVE™ Modality Overview
AUTHORS: Nathan Heath Patterson, Elizabeth Neumann, Christine Surrette, Soumya Ghose, Liz McDonough, Jamie Allen, Jeff Spraggins, Fiona Ginty

[DESCRIPTION]
This is an overview of all protocols currently in use for the GE/Vanderbilt University Cell DIVE collaboration for the Human BioMolecular Atlas Program (HuBMAP). It includes links to each of the individual protocols that make up this project workflow.

[STEPS]
SECTION: MALDI IMS
5. Perform Matrix Application

 


SECTION: Cell DIVE
9. Characterize antibodies (primary/secondary, direct conjugates, and zenon labelled antibodies) and determine any antigen effects from the Cell DIVE dye inactivation process.


Cell DIVE™ Platform | Antibody Characterization for Multiplexing 
Cell DIVE™ Platform | Antibody Staining & Imaging

SECTION: Cell DIVE
10. Prepare direct conjugates for study.

Cell DIVE™ Platform | Antibody Purification Chemistry
Cell DIVE™ Platform | Ab Conjugation: Initial Conjugation & Scale up Conjugation
SECTION: Cell DIVE
11. Perform Cell DIVE™ multiplexed data acquisition on the final cohort. 

 

SECTION: MALDI IMS
1. Collection of post-surgical tissue.
 

SECTION: MALDI IMS
2. Stabilize and freeze tissues.
 

SECTION: MALDI IMS
6. Perform high resolution IMS analysis of matrix coated tissue sections.
 


SECTION: Preparing Sample for MxIF
8. Perform Matrix Removal & Tissue Fixation

 

SECTION: MALDI IMS
3. 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.
 

SECTION: MALDI IMS
4. Perform autofluorescence microscopy on all tissue sections 
 

6. Perform fluorescence microscopy to visualize laser ablation spots.
Post IMS AF: 
 

10. 
7. 
9. 
SECTION: MALDI IMS
9. Obtain autofluorescence microscopy images of tissues after IMS analysis
 

SECTION: MALDI IMS
7. Obtain autofluorescence microscopy images of tissues after IMS analysis
TITLE: Ego-team
AUTHORS: Mantilla Cesar, Zahra  Murad

[DESCRIPTION]
We study how individuals' contribution to a team production task varies depending on whether the task is ego relevant or not. 
We design and conduct an experiment to test the effect of ego-relevance when the team output depends on the top- and the bottom-performer of the group.
Ego-relevance is manipulated by calling the Raven IQ Test an “IQ Task” or a “Pattern Task”. 
We find that the contribution, which corresponds to an contribution of intended effort in the task, is affected by ego-relevance and the team production function.
However, both effects are mediated by the expected teammate's contribution. 
Ego-relevance increases the responsiveness to the expected teammate's behavior, a behavior that is also more noticeable when the team output depends on the bottom-performer.
Nevertheless, we do not observe crossed-effects between ego-relevance and the team production function.

[STEPS]
TITLE: EXPERIMENTO MAGNETISMO
AUTHORS: Keven Alexis Aispuro Bueno, Abraham Espinoza Cazares, Diego Aleiandro Malo Osuna, Eduardo Madrigal, a

[DESCRIPTION]
.

[STEPS]
SECTION: Preparación del experimento
1. Primero, prensamos una agujeta de zapato con 5 imanes por un extremo.

SECTION: Preparación del experimento
2. Por otro extremo le pegamos un pedazo de cinta con el objetivo de poder medir la posición respecto al tiempo.
SECTION: Preparación del experimento
3. Pegamos un tubo de cobre de 1 metro a una pared con cinta azul.
SECTION: Realización del experimento
4. Introducimos el cordón bien extendido, que el extremo con cinta salga por el otro lado del tubo.
SECTION: Realización del experimento
5. Dejamos caer los imanes y grabamos el recorrido del pedazo de cinta.
SECTION: Recolección de datos
6. Con ayuda de tracker tomas medidas del video.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the protocol to be used in the quantification of glial fibrillary acidic protein (GFAP) in mouse brain tissue. </p>
<p><strong> </strong></p>

[GUIDELINES]
<p><strong>TRAINING REQUIREMENTS</strong></p>
<p>Able to use a plate reader and to accurately dilute tissue samples.</p>
<p> </p>
<p><strong>REFERENCES</strong></p>
<p>Paper<em>: Quantification of Glial Fibrillary Acidic Protein: Comparison of Slot-Immunobinding Assay with a Novel Sandwich  ELISA</em>, O’Callaghan, J.P., Neurotoxicology and Teratology, Vol. 13, pp. 275-281, Pergamon Press plc. 1991</p>
<p> </p>
<p>SoftMax User’s Manual, Molecular Devices Corporation, Sunnyvale, CA</p>

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol we are using to assess the Light/Dark preference in flies.

[BEFORE_START]
The light source should be homogenous above the T-Maze.

[GUIDELINES]
<p>The Choice Index is calculated using the formula:<br /><br /></p>
<p>CI=((#FL&times;1)+(#FD&times;-1)+(#FE&times;0))/(#FT )</p>
<p><br />where #FL, #FD, #FE, and #FT mean the number of flies in the transparent tube, the opaque tube, the number of flies that remained in the elevator, and the total number of flies, respectively. A&nbsp;<em>CI</em>&nbsp;of 1 mean all the flies chose the light, while an index of -1 mean a negative phototaxis and 0 meant no choice. In each experiment a CI is calculated for the wingless flies and other for the intact flies.</p>
<p><a href="http://lab.brembs.net/wp/wp-content/uploads/2015/02/TMaze-1.png" target="_blank"><img class="alignnone size-medium wp-image-1203" src="http://lab.brembs.net/wp/wp-content/uploads/2015/02/TMaze-1-212x300.png" alt="TMaze 1" width="212" height="300" /></a></p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div>
<p>This protocol is from 'Flower Ca<sup>2+</sup> channel in CME and ADBE' of Yao CK et al.</p>
<div>
<div>
<p> </p>
<p>Please see the manuscript for the full method details.</p>
</div>
</div>
</div>

[BEFORE_START]
<p>You'll need: </p>
<p> </p>
<p><strong>0 mM Ca<sup>2+</sup> hemolymph-like (HL)-3 solution:</strong></p>
<ul>
<li>70 mM NaCl</li>
<li>5 mM KCl</li>
<li>10 mM MgCl<sub>2</sub></li>
<li>10 mM NaHCO<sub>3</sub></li>
<li>5 mM trehalose</li>
<li>5 mM HEPES (pH 7.2) </li>
<li>115 mM sucrose</li>
</ul>
<p> </p>
<p><strong>90 mM K<sup>+</sup>/0.5 mM Ca<sup>2+</sup> stimulation:</strong></p>
<p>(or alternative 60mM K<sup>+</sup>/1mM Ca<sup>2+</sup>)</p>
<ul>
<li>25 mM NaCl</li>
<li>90 mM KCl</li>
<li>10 mM MgCl<sub>2</sub></li>
<li>10 mM NaHCO<sub>3</sub></li>
<li>5 mM trehalose</li>
<li>5 mM HEPES (pH 7.2)</li>
<li>30 mM sucrose</li>
<li>0.5 mM CaCl<sub>2</sub></li>
</ul>
<p><strong>solution of 90 mM K<sup>+</sup>/2 mM Ca<sup>2+</sup>/200 mM chlorpromazine:</strong></p>
<ul>
<li>25 mM NaCl</li>
<li>90 mM KCl</li>
<li>10 mM MgCl<sub>2</sub></li>
<li>10 mM NaHCO<sub>3</sub></li>
<li>5 mM trehalose</li>
<li>5 mM HEPES (pH 7.2)</li>
<li>30 mM sucrose</li>
<li>2 mM CaCl<sub>2</sub> </li>
<li>200 mM chlorpromazine</li>
</ul>
<p> </p>

[STEPS]
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TITLE: Cleaning up a biohazardous spill inside a centrifuge
AUTHORS: Steven Wilhelm, Gary LeCleir, Ashley Humphrey

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">BIOLOGICAL SPILL RESPONSE </span><span>method to be used in the event of a biosafety incident. </span></div></div>

[STEPS]
?. Close off the area and allow aerosols to settle.
?. Notify others including supervisor.
?. Wait  to allow the aerosols to settle.
?. Don appropriate PPE: laboratory coat, safety glasses and Nitrile gloves.
?. Remove rotors and bucket and place in Biosafety Cabinet.
?. Thoroughly disinfect the inside and outside of the centrifuge rotor, cups and accessories and allow proper contact time. After disinfection, move to sink for a thorough rinse, dry thoroughly.
?. For the centrifuge interior, follow the steps above for cleaning up a spill outside a Biosafety Cabinet.
?. Segregate contaminated cleanup materials into the appropriate biohazardous waste containers.
TITLE: Western Blotting (Fly Heads)
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol describes how to perform a west blotting technique using fly heads.

[STEPS]
1. Homogenize desired number of fly heads in 1 X Laemmli sample buffer.
2. Heat samples to 100 °C for 10 min , spin briefly before loading.

3. Load premade gel into western blotting apparatus. Fill reservoir with Running Buffer:
Running Buffer:
6 g  Tris-HCL
28.9 g  glycine
Fill to 1 L  with distilled water
Add 5 mL  20% SDS

4. Load samples on gel and attach electrodes.
5. Run gel at 120 V until dye front reaches the bottom of the gel, 60 min .Run longer for greater separation.

6. Remove gel and transfer using Trans-Blot Turbo.
7. Perform antigen retrieval by microwaving 9 min  in PBS.

8. Block membrane in 1X PBS with 0.05% Tween-20 and 3% dry milk for 60 min .

10. Wash blot 3x in PBSTween, 5 min each, with shaking.

11. Add secondary antibody at the correct dilution in PBSTween + milk, incubate with shaking at Room temperature  for 180 min .

12. Wash blot in PBSTween 30 min  with frequent wash changes.

9. Add primary antibody at correct dilution in PBSTween + milk and incubate with shaking    at 4 °C .

13. Develop with ECL substrate or image fluorescence, as appropriate.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>

[GUIDELINES]
<p><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/kdeeuxn.jpg" width="479" height="441" data-src="https://s3.amazonaws.com/pr-journal/kddeuxn.jpg" data-ofn="IMG_4447.jpg" /></p>
<p> </p>
<p>A: M12 System</p>
<p>B: Filter cartridge (refrigerated while stored and supported by a clamp during use)</p>
<p>C: Sample water </p>
<p>D: Retentate</p>
<p>E: Permeate</p>
<p>F: Control panel</p>
<p>G: Tygon tubing</p>

[STEPS]
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TITLE: Protein Lysate Preparation Protocol (Tissue)
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details the preparation of protein lysate of the tissue sample.


[STEPS]
SECTION: Protein Lysate Preparation (Tissue)
1. If tissue stored at -80 °C, place in -20 °C for at least 240 min or   prior to homogenization. 

SECTION: Protein Lysate Preparation (Tissue)
2. Weigh tissue out in mg.
SECTION: Protein Lysate Preparation (Tissue)
3. Add in 10 volumes of 1X TNE (e.g. 10 µL of TNE per 1 mgof tissue).
SECTION: Protein Lysate Preparation (Tissue)
4. Either by mechanical (Dounce) or homogenizer machine, homogenize tissue gently and on ice.

SECTION: Protein Lysate Preparation (Tissue)
5. This is TNE crude lysate (no detergents).
SECTION: Protein Lysate Preparation (Tissue)
6. Add equal volume of 1xTNE +1% SDS, 0.5% NP-40, and 0.5% Doc (Complete TNE).
SECTION: Protein Lysate Preparation (Tissue)
7. Sonicate at 40 °C (3 pulses: 10s On/2s Off at 20% amplitude).
SECTION: Protein Lysate Preparation (Tissue)
8. Boil (10 min).


 

SECTION: Protein Lysate Preparation (Tissue)
9. Centrifuge (16000 x g, 10 min, 4 °C).

SECTION: Protein Lysate Preparation (Tissue)
10. Collect supernatant for BCA protein assay.
TITLE: Library Aligner and NGS Cas9 Cutting Analysis
AUTHORS: Colin Kremitzki, William J Buchser

[DESCRIPTION]
Protocol on how to run Library Aligner in FIVTools once sequencing data has been returned to us from the Sequencing Core.  It also has an option for checking the Representation of a gRNA library.  Lastly, a video is posted on how to  check your NGS reads for Cas9 cutting analysis.

[STEPS]
1. Open FIVTools and click on the LA tab in the upper left section of the program.

 
2. Enter in your returned NGS link into the HTCF Link tab and the destination to download the files into Dest.

 
3. Press Download UZ to download the files and automatically unzip them.  This will deposit reads into your destination folder inside folders called FastQ and FastQ_gz.
4. Once downloaded, you can press the button Defaults under XML Settings: to return the left criteria back to the defaults.
5. In the left tab for PathReferenceList, enter in the pathway which contains the reference .txt file.  This file should contain the gRNA sequences that you wish to align to your reads.  If you wish to align to multiple different .txt files, just list the directory and not a specific file.
6. In the left tab for PathFastQToSearch, enter in the pathway to your FastQ files.

 
7. The rest of the settings can be adjusted to fit your schema. 
8. Press Start once you are ready to align your reads to your reference sequence list.
9. When LA is running, you will see several things happen on your screen.  You will want to be looking for the % of Unmapped Reads.  If this is 100%, then your reads are not matching anything in your reference list.  If you have a low % of Unmapped Reads, then that means they are hitting to reference reads, and that’s a good thing.
10. Once finished, LA will deposit a lot of files in your destination folder.  FastA files, ref_f.txt (what most people will want to look at - includes a counts table), ref_long.txt (fancier version used in Spotfire analysis), ref_TopSeq.txt (good for looking at your top hits if you get a low number of matches - answers the question: what are you matching to?).

 
11. If you wish to check the Representation of the gRNA libraries you’ve put into cells, you can do this at the bottom of Library Aligner.

 
12. Enter in the MOI you used to infect your cell line, the titer, and the Cell Factor.  Cell Factor is 1 if you were using hearty cells like U2OS or HeLa cells.  Cell Factor will be 2 if you were using delicate cells like iPSC cells. Select Merge P if you wish to merge SubPools together in the Representation Check, ie. MSPH1 through MSPH7 will be joined together.  Press Check Rep when ready.
13. You can also check your representation via the following website:  https://libalignerminiature20221013181154.azurewebsites.net/
14. Willie also created a youtube video on how to utilize FIVTools to check for Cas9 transfected gRNA cutting.  That video can be found here:  https://www.youtube.com/watch?v=b2_2suSgahg&list=PLHBFVqDRJN5VuV9CPXAfiiwMpZVOquSx_&index=35
TITLE: Temae Golf Course Surveys
AUTHORS: Kedhar Bartlett, Bridget Bentley

[DESCRIPTION]
This study investigates the ecological impacts of land use change, particularly focusing on the vegetation near the Temae Golf Course on Moorea, French Polynesia. Through field surveys conducted by the 2024 Island Sustainability Program cohort, we compare plant diversity and composition in pristine and cultivated areas adjacent to the golf course. Our findings highlight significant differences, with cultivated areas exhibiting lower diversity and a higher proportion of introduced species. These results underscore the potential risks associated with tourism-driven land development, including the introduction of invasive species and the loss of ecosystem resilience. By shedding light on these impacts, our study emphasizes the importance of integrating conservation efforts with tourism development to ensure the long-term sustainability of Moorea’s ecosystems.

[GUIDELINES]
Avoid trespassing on private property; if care is not taken, you may end up on golf course property or local backyards.

[STEPS]
SECTION: Field Methods
1. Split group into pairs
SECTION: Field Methods
1.1. Assign each pair a "cultivated" (on/near golf course) site and a "pristine" (anywhere else in close proximity) site
SECTION: Field Methods
2. Conduct 50 meter transects at each site
SECTION: Field Methods
2.1. Note starting point and direction of movement in field notebook, and walk in a straight line in that direction for the duration of the transect
SECTION: Field Methods
2.2. One group member measures distance, and the other photographs plant species. Both group members look for new plant species within 1 meter of each side of the transect line. 
SECTION: Field Methods
2.3. If unable to complete the full 50m transect, note that and mention why
SECTION: Field Methods
2.4. Repeat at other site (either cultivated or pristine, whichever you haven't already completed)
SECTION: After fieldwork
3. Identify plants from photos taken in the field
SECTION: After fieldwork
3.1. Groups either used available textbooks or iNaturalist
SECTION: After fieldwork
4. Compile all pairs' data in a common Google Sheet (linked here)