eamag/protocols-with-steps huggingface dataset:
TITLE:  Enzyme linked immunosorbent assay (ELISA) for determining the plasma or serum concentration of IL-33 in humans.
AUTHORS: Angel  Justiz-Vaillant

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

[STEPS]
?. Ninety-six well ELISA plates are coated with monoclonal anti-human antibodies to interleukin-33 (IL-33).
?. Patient serum samples are added to the plates.
?. The plate is incubate x 1.30 hour at RT.
?. The plate is washed 4 times with PBS-tween buffer.
?. The wells are incubated with a biotin conjugated anti-human IL-33 for 1.30 hour at RT.
?. The plate is washed again as above.
?. Add to the plate a peroxidase-labeled streptavidin conjugate and incubate it for 1 hour at RT.
?. After a further washing procedure a substrate solution reactive is added and allowed to produced a colored reaction in positive controls.
?. The level of IL-33 in the sample is proportional to the colored product developed.
?. The addition of 3M H2SO4 stops the reaction.
?. The absorbance is measured at 450 nm.
?. The IL-33 concentration can be calculated by generating an standard curve using lineal regression.
TITLE: Anchorage-independent growth assay or Soft Agar assay
AUTHORS: Marzia Ognibene

[STEPS]
?. Prepare 5% SeaKem low gelling agarose in distilled water and autoclave it.
?. Warm 10% serum media in 48 °C water bath
?. Melt the agar in microwave for some minutes. Be sure to mark the level of agar prior to heating so the level can be brought back up with distilled water, to maintain the correct concentration of agar.
?. Allow the agar to cool at room temperature for 10 minutes, then place it in the 48 °C water bath.
?. Make a dilution of agar in prewarmed media for a final concentration of 0.6% agar and considering to pour 4 ml in each p60 plastic plates.
?. Pour the agar in plates and let them sit at room temperature for 40 minutes to solidify, then place in the 37 °C CO2 incubator to allow the plates to adjust to the proper pH.
?. Prepare a dilution of agar in prewarmed media for a final concentration of 0.3% agar and considering to pour 3 ml in each p60 plastic plates.
?. Trypsinize cells and resuspend in a minimal volume of media to count them and make the desired cell dilution.
?. Add 3 ml of 0.3% agar into appropriate number of conical tubes.
?. Add the right amount of cells to each tube and, working quickly, gently shake the tube in order to suspend the cells evenly through the agar.
?. Pour the entire content of tube on top of a previously poured 0.6% solidified agar plate.
?. Swirl plates gently and let them sit at room temperature for 40 minutes to solidify, then place in the 37 °C CO2 incubator.
?. After 24 hours, feed cells with 1 ml of 10% serum media and check weekly that they are well hydrated.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Background: Until recently many researchers have examined energy and fatigue as opposite ends of a bipolar spectrum rather than two separate unipolar moods. Studies have also focused on a single variable to study these mood states rather than examining multiple variables simultaneously. Therefore, the purpose of this study was to identify factors predicting feelings of energy and fatigue while simultaneously examining multiple domains related to these mood states in graduate health sciences students.
Methods: Seventy-seven subjects were recruited from a Physician Assistant, Physical Therapy and Occupational Therapy program at a small school in Northern New York. Subjects completed a series of surveys to measure mood, diet, mental work load intensity on school days and non-school days, and physical activity. Subjects also completed the Trail-making Test Part B task on an iPad and their Resting Metabolic Rate (RMR) and muscle oxygen consumption mVO2 was measured. A backwards linear regression was used to determine the relationship between energy, fatigue and multiple variables.
Results: The predictor variables accounted for 46.1% and 22.7% of the variance in fatigue and energy, respectively. More fatigue was associated with worse sleep quality, more time spent sitting and higher perceived intensity of mental workload on non-school days. More energy was associated with better sleep quality, higher muscle oxygen saturation, lower RMR, and faster psychomotor performance. 
Conclusion: The results of this study indicate that energy and fatigue are separate constructs that are predicted with different accuracy by different variables. Our results indicate that small lifestyle changes may be necessary to improve feelings of fatigue but comprehensive interventions may be necessary to improve feelings of energy. This study provides new insight into a multi-domain approach to predicting energy and fatigue. 

[STEPS]
?. 
SECTION: Measure weight and body composition using the Tanita TBF-410-GS
?. 
SECTION: Measure height using a standard stadiometer
?. 
SECTION: Complete surveys
?. 
SECTION: Complete mental energy test
?. 
SECTION: Measure Resting Metabolic Rate (RMR) and muscle oxygen saturation
?.
TITLE: A simple method for purification of immunoglobulin-Y (IgY).
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. Separate egg yolk from the egg white.
?. Add ten parts of distilled water to one part egg yolk.
?. Mix and stir slowly for 6.30 h at 4°C.
?. Centrifuge at 10 000 × g, at 4°C for 27 min to precipitate the lipids.
?. Collect the supernatant containing the IgY.
?. Slowly add potassium sulfate to the preparation or specimen, stirring constantly, to a ﬁnal concentration of 500 mM.
?. Adjust pH to 7.6.
?. Pass the sample through a 0.45 µm ﬁlter just before applying it to the HiTrap™ Column column.
TITLE: DAB Staining
AUTHORS: daniel.dautan, Per Svenningsson

[DESCRIPTION]
3, 3'-diaminobenzidine (DAB) staining of mouse brain tissue

[GUIDELINES]
All steps should be done on an orbital shaker.

[STEPS]
SECTION: Staining/Mounting Process
1. Wash freshly sectioned slices of tissue 2-3 times with 1X PBS to remove OCT. 
SECTION: Staining/Mounting Process
2. Quench sections for 15 min in 3 mL of quenching solution (0.1 mL 30% H2O2, 0.1 mL methanol, and 0.8 mL 1X PBS).
SECTION: Staining/Mounting Process
3. Wash 4-5 times in 1X PBS. 
SECTION: Staining/Mounting Process
4. Block sections in 2 mL of 5% goat serum in 0.25% T-PBS for 60 min Room temperature.
SECTION: Staining/Mounting Process
5. Stain with primary antibody (pSer129 primary antibody 1:500) overnight at 4 °C. Dilute primary antibody in 2.5% serum in 0.25% T-PBS (1 mL per well).
SECTION: Staining/Mounting Process
6. Wash sections 4-5 times with 1X PBS.
SECTION: Staining/Mounting Process
7.  Transferred sections into secondary solution (1 mL of 1% serum in 0.25% T-PBS) for 120 min at Room temperature. 

SECTION: Staining/Mounting Process
8. Wash sections 4-5 times in 1X PBS. 
SECTION: Staining/Mounting Process
10. Wash sections 4-5 times in 1X PBS. 



SECTION: Staining/Mounting Process
11. Transfer sections to the DAB working solution (SK-4100, Vector Laboratories) under a fume hood. Monitor until it stains well. Well-plate containing sections were gently shaken during staining and the reaction was stopped with transfer to 1X PBS based on dark-signal intensity on the fastest arising staining.
SECTION: Staining/Mounting Process
12. Wash sections 3-5 times in 1X PBS.
SECTION: Staining/Mounting Process
13. Mount sections on microscope slides. 
SECTION: Staining/Mounting Process
14. Dry sections 60 min at Room temperature.

16. Wash with 70% ethanol (2 times ~2 min).

17. Wash with 95% ethanol (2 times ~2 min).

18. Wash with 100% ethanol (2 times ~2 min).

19. Wash with 100% xylene (2 times ~5 min) to allow section dehydration. 

SECTION: Staining/Mounting Process
9. Transfer into ABC Kit solution (PK4000, Vector Laboratories) containing 10µl of solution A and 10µl of solution B for 1ml of 1X PBS for 60 min at Room temperature. 
15. Dehydrate sections starting with 2 baths of distilled water (~2 min).

20. Dry sections at Room temperature (~5 min) and covered with DPX mounting medium.
TITLE: Automation, live-cell imaging, and endpoint cell viability for 96-well plate drug screens
AUTHORS: Rolando DZ Lyles, M Julia Martinez, Benjamin Sherman, Kerry Burnstein

[DESCRIPTION]
To streamline the identification of potentially active cancer therapeutics, here we describe a highly adaptable semi-automated approach to screen compounds simultaneously across a panel of cell lines. These protocols leverage automation to enhance robustness, reproducibility, and throughput while integrating the IncuCyte ZOOM live-cell imaging platform and the CellTiter-Glo endpoint viability assay to assess drug efficacy. The integration of both evaluative methods strategically bypasses the shortcomings of each approach individually allowing for more thorough and detailed analysis within a single drug screen. The expected output from protocol utilization includes traditional dose-response curves, IC50, area-under the curve (AUC), GR50, area-over the curve (AOC) as well as live-cell imaging to identify cell specific morphological changes, cytostatic, or cytotoxic effects of 72-hour drug treatments. This adaptable protocol can be employed across cancer model systems and represents a reproducible procedure to optimize 96-well plate cell growth conditions compatible with the integrated drug screen and simultaneously assess drug efficacy across multiple cell lines in future cancer research studies.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>The ROS measurements of the supernatant is performed by using DCF-DA, 2’,7’- dichlorofluorescein diacetate, a fluorescent probe for the assay. The formation of the oxidized fluorescent derivative (DCF) is monitored at excitation and emission wavelengths of 488 and 525 nm, respectively, in a fluorescent plate reader.</p>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>DNA-Blot genotyping of </strong><strong><em>Trypanosoma cruzi </em></strong><strong>Discrete Typing Units (DTU) with radiolabeled  probes</strong></p>

[BEFORE_START]
<p><span style="font-weight: 400;">1. Prepare solutions:</span></p>
<ul>
<li style="font-weight: 400;"><span style="font-weight: 400;">EDTA 0.5M, pH8.</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">TBE 10X: Tris 0.89M + Boric Acid 0.89M+ EDTA 0.5M 40 ml + distilled H</span><span style="font-weight: 400;">2</span><span style="font-weight: 400;">O to complete 1000 ml</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">TBE 1X:Tris (0.089M) - Borate (0.089M) - EDTA (0.002M). Use 100 ml of TBE 10X + 900 ml distilled H</span><span style="font-weight: 400;">2</span><span style="font-weight: 400;">O.</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Denaturation solution:  NaCl 1.5M NaOH 0.5M</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Neutralization solution: Tris 1M pH 7.4 NaCl 1.5M</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">Hybridization solution SSC 20X: NaCl 3M Na</span><span style="font-weight: 400;">3</span><span style="font-weight: 400;">C</span><span style="font-weight: 400;">6</span><span style="font-weight: 400;">H</span><span style="font-weight: 400;">5</span><span style="font-weight: 400;">O</span><span style="font-weight: 400;">7</span><span style="font-weight: 400;"> 0.3M pH7</span></li>
<li style="font-weight: 400;"><span style="font-weight: 400;">SSC 2X: Use 50 ml of SSC 20X + distilled H</span><span style="font-weight: 400;">2</span><span style="font-weight: 400;">O to complete 500 ml.</span></li>
</ul>
<p> </p>
<p><span style="font-weight: 400;">2. Amplify </span><em><span style="font-weight: 400;">Trypanosoma cruzi</span></em><span style="font-weight: 400;">’s kinetoplastidic DNA by PCR using primers 121 and 122 (Wincker P, Britto C, Pereira JB, Cardoso MA, Oelemann W, Morel CM. Use of a simplified polymerase chain reaction procedure to detect </span><em><span style="font-weight: 400;">Trypanosoma cruzi</span></em><span style="font-weight: 400;"> in blood samples from chronic chagasic patients in a rural endemic area. Am J Trop Med Hyg. 1994;51(6):771-777). </span></p>

[GUIDELINES]
<ul>
<li><span style="font-weight: 400;">Use gloves, mask and eye protection to prepare solutions.</span></li>
<li><span style="font-weight: 400;">Use gloves during the whole procedure.</span></li>
</ul>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunohistochemistry for p-eIF2a and eIF2a, v.230804
AUTHORS: elanalockshin

[DESCRIPTION]
Authors:
Callie Eatman, Elana Lockshin with recommendations from Xinlu Ding, Dan lab, UC-Berkeley
Calakos lab
Duke University



[STEPS]
SECTION: Reagents
1. 

phospho-eIF2a antibody: Abcam ab32157


eIF2a antibody: Cell signaling #5324



SECTION: Tissue Prep
2. Brains are stored in 4% PFA for 24hrs in 4C before storage in 1xPBS in 4C until sectioning
Brain is sectioned in 1xPBS bath into 40um slices using Vibratome.
Slices are stored in 0.03% Sodium Azide in 1xPBS in 24 well plates in 4C.
SECTION: IHC
3. In 24 well plate/ 2 slices per well with a minimum of 300uL of solution in well
SECTION: IHC
4. Day 1:
1. Alkaline Retrieval: 20 min in 1% NaOH (in 1x PBS) on shaker (RT) (make fresh each time, do not store in glass)
SECTION: IHC
5. 2. 3x10 min wash on shaker in PBS-T (PBS with 0.3% Triton X-100) (RT)
SECTION: IHC
6. 3. 2 hr blocking in 10% NDS/1% BSA in PBS-T (RT)
SECTION: IHC
7. 4. Primary antibody in dilution buffer (5% NDS +1%BSA) in PBS-T 48hrs in 4C on shaker
a. P-eIF2a Antibody (dilution 1:100 )
OR
b. eIF2a Antibody (dilution: 1:500 )
Day 2:
SECTION: IHC
8. Day 2:
1. 3x10 min wash on shaker in PBS-T (RT)
SECTION: IHC
9. 2. Secondaries for 2hrs in dilution buffer (5% NDS +1%BSA) in PBS-T (RT)
● DK~Rb Alexa fluor 488+ (1:500)
SECTION: IHC
10. 3. 3x10 min wash on shaker in PBS-T (RT)
○ (for Hoescht: 1:750 in 1st 10 min wash)

SECTION: IHC
11. 4. Mount on slides by floating section in PBS and let sit until dry
SECTION: IHC
12. 5. Coverslip with Vectashield hardset mounting medium
SECTION: IHC
13. 6. Seal slide with nail polish next day
SECTION: IHC
14. 7. Store in 4C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<h3>Antibiotic Concentrations</h3>
<table>
<thead>
<tr>
<th>Antibiotic</th>
<th>Recommended Stock Concentration</th>
<th>Recommended Working Concentration</th>
</tr>
</thead>
<tbody>
<tr>
<td>Ampicillin</td>
<td>100 mg/mL</td>
<td>100 µg/mL</td>
</tr>
<tr>
<td>Bleocin</td>
<td>5 mg/mL</td>
<td>5 µg/mL</td>
</tr>
<tr>
<td>Carbenicillin*</td>
<td>100 mg/mL</td>
<td>100 µg/mL</td>
</tr>
<tr>
<td>Chloramphenicol</td>
<td>25 mg/mL<br />(dissolve in EtOH)</td>
<td>25 µg/mL</td>
</tr>
<tr>
<td>Coumermycin</td>
<td>25 mg/mL<br />(dissolve in DMSO)</td>
<td>25 µg/mL</td>
</tr>
<tr>
<td>Gentamycin</td>
<td>10 mg/mL</td>
<td>10 µg/mL</td>
</tr>
<tr>
<td>Kanamycin</td>
<td>50 mg/mL</td>
<td>50 µg/mL</td>
</tr>
<tr>
<td>Spectinomycin</td>
<td>50 mg/mL</td>
<td>50 µg/mL</td>
</tr>
<tr>
<td>Tetracycline</td>
<td>10 mg/mL</td>
<td>10 µg/mL</td>
</tr>
</tbody>
</table>
<p><em><strong>Note:</strong> Unless otherwise indicated, the antibiotic powder can be dissolved in dH<sub>2</sub>O.</em></p>
<p><em><strong>*Note:</strong> Carbenicillin can be used in place of ampicillin. Carbenicillin is more stable, so it is potentially more effective at selecting only bacteria containing the plasmids of interest (for example, fewer satellite colonies will grow). However, it is also more expensive.</em></p>

[STEPS]
?.
TITLE: Immunohistochemistry 
AUTHORS: Miguel Chuapoco

[DESCRIPTION]
Outlines procedures to perform immunohistochemistry on tissue that had been treated with AAV. 

[STEPS]
SECTION: Tissue slicing via vibratome (mice and rhesus macaque)
1. All tissue should be stored in PBS prior to slicing and further processing. Remove tissue and prepare for mounting. For mouse brain, we separate each half hemisphere. Rhesus macaque brain was separated into 4 mm thick coronal blocks. For all other tissue (e.g. liver), remove a 4-5 mm piece of tissue and cut one edge to make a flat surface. 
SECTION: Tissue slicing via vibratome (mice and rhesus macaque)
2. Use super glue to mount the tissue on the provided mounting disk and fill reservoir with 1X PBS. 
 

SECTION: Tissue slicing via cryostat (marmoset and rhesus macaque)
4. All tissue should be stored in PBS prior to slicing and further processing. Remove tissue and incubate in 30% sucrose at 4 °C on a shaker. For marmoset tissue, incubate overnight. For rhesus macaque tissue, incubate for 72 hours, changing the 30% sucrose solution after 48 hours. 

SECTION: Tissue slicing via vibratome (mice and rhesus macaque)
3. Slice tissue to 50-100 um slice and collect in 24- (mouse) or 6- (rhesus macaque) well plates filled with 1X PBS. 

Tissue can also be cut to 300 um to be used for index refraction matching (e.g. to perform morphological reconstructions). 
SECTION: Tissue slicing via cryostat (marmoset and rhesus macaque)
5. Embed tissue in OCT using 100% isopropanol (or similar) chilled with dry ice. Orient tissue to ensure coronal slices. 

   

 

 

 

 

SECTION: Tissue slicing via cryostat (marmoset and rhesus macaque)
6. Store tissue at -80 °C overnight before continuing to slicing. 

SECTION: Tissue slicing via cryostat (marmoset and rhesus macaque)
7. Cut tissue to 50 um slice and collect in 24- (marmoset) or 6- (rhesus macaque) well plates filled with 1X PBS. 
SECTION: Immunohistochemistry
8. Incubate slices in primary antibody overnight on a shaker at room temperature. Dilute antibody in 1X PBS supplemented with 0.1% Triton X-100   and 10% normal donkey serum  . 

 

SECTION: Immunohistochemistry
9. Wash with PBS 3-5 times at room temperature on a shaker.
SECTION: Immunohistochemistry
10. Incubate slices in secondary antibody overnight on a shaker at room temperature. Dilute antibody in 1X PBS supplemented with 0.1% Triton X-100 and 10% normal donkey serum. 

Protect from light to avoid photobleaching.


 

SECTION: Immunohistochemistry
11. Wash with PBS 3-5 times at room temperature on a shaker.

Protect from light to avoid photobleaching.

SECTION: Immunohistochemistry
12. Mount sections on slides and allow slice to dry at room temperature in the dark.  Mount coverslip using Prolong Diamond Antifade.
TITLE: PIP MRI Magnetization Transfer Ratio (MTR) Measurement of Myelofibrosis in Mouse Tibia
AUTHORS: Thomas L Chenevert

[DESCRIPTION]
The goal of this Co-Clinical Imaging Research Program (CIRP) pre-clinical imaging protocol (PIP) is to provide detailed description of key steps used to achieve a stated level of technical repeatability (precision) embodied in “Claims”, for MRI measurement of magnetization transfer ratio (MTR) in tibia bone marrow of myelofibrosis mouse models. This pre-clinical imaging procedure document will be referred to as a “profile” and adheres to a PIP MRI template provided in: https://www.protocols.io/workspaces/pre-clinical-imaging-protocols. 
Here we treat MTR as a “quantitative” metric, although numerical MTR results are a strong function of multiple experimental parameters and MRI hardware conditions. This profile will adopt the common practice to express MTR values in on a 0 to 1 scale. Empirically, MTR reflects fraction of visible signal loss due to application of off-resonance saturation RF energy depleting magnetization in the semi-solid (invisible) macromolecular matrix thus reducing magnetization replenishing visible signal.Simple fluids free of macromolecules should have an MTR » 0, whereas macromolecule-rich semi-solid tissues such as myelin, muscle, scar and collagen exhibit relatively high MTR (> 0.5). This document details procedures for MTR measurement in MF mouse tibia to achieve stated performance claims.
Tibia bone marrow composition in MF mouse models has gradation going from proximal to distil ends of the tibia, therefore separate claims are made for volume of interest (VOI) analysis of ADC maps for each of three distinct sections along the length of the tibia (see Figure 1):
Section 1 (proximal)º VOI (~4-5mm3) within 9mm of proximal end of tibia
Section 2 (transition)º VOI (~0.4-0.5mm3) from 10 to 12mm of proximal end of tibia
Section 3 (distil)º VOI (~0.1-0.2mm3) from 13 to 14mm of proximal end of tibia

Claim 1:A measured change in the mean ADC in Section 1 VOI of MF mouse model tibia that exceeds ±0.037µm2/ms indicates a true biological change has occurred in the tibia bone marrow with 95% confidence.
Claim 2:A measured change in the mean ADC in Section 2 VOI of MF mouse model tibia that exceeds ±0.087µm2/ms indicates a true biological change has occurred in the tibia bone marrow with 95% confidence
Claim 3:A measured change in the mean ADC in Section 3 VOI of MF mouse model tibia that exceeds ±0.030µm2/ms indicates a true biological change has occurred in the tibia bone marrow with 95% confidence

[STEPS]
TITLE: Prepare NGM plates for nematode, with peptone, without fungizone
AUTHORS: Cancer Research UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prepare NGM plates for nematodes, with peptone, without fungizone</div></div>

[STEPS]
?. 
?. ABC1Ingredients Quantity2NaCl 3g3bacto peptone 2.5g4Agar 17g5Double
  distilled H2O972ml
ABC1Ingredients Quantity2NaCl 3g3bacto peptone 2.5g4Agar 17g5Double
  distilled H2O972ml
?. A1Measure 972ml&nbsp;
  double distilled H2O and put in a 1L duran bottle with a magnetic flea2Add 3g NaCl, 2.5g bacto peptone and stir.3Stir until all solutes are dissolved then add the
  agar.4Leave magnetic flea in bottle.5Label, date and autoclave
A1Measure 972ml&nbsp;
  double distilled H2O and put in a 1L duran bottle with a magnetic flea2Add 3g NaCl, 2.5g bacto peptone and stir.3Stir until all solutes are dissolved then add the
  agar.4Leave magnetic flea in bottle.5Label, date and autoclave