eamag/protocols-with-steps huggingface dataset:
TITLE: Chimeric Protein-LAG and Protein LA sandwich ELISA 
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between protein-LAG (PLAG) and protein-LA (SpLA) with different immunoglobulin preparations from mammalian and avian species.</div></div>

[STEPS]
?. This ELISA was used to study the interactions between protein-LAG (PLAG) and protein-LA (SpLA) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of PLAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpLA conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.32.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Sanger sequencing was performed on available samples from all affected family members to determine whether the potential mutation in the causative gene co-segregated with the disease phenotype. In order to amplify exon 7 of the <em>ILDR1</em> gene, we designed the following primers: ILDR1-7F: TTGATGTCCTGATTCTGAGG and ILDR1-7R: CTCTGTGGTGGAATGAGAGG. The amplified products were then purified using Wizard SV Gel and PCR Clean-up system (Promega, USA) and were consequently sequenced using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Thermo Fisher Scientific, USA). The resulting sequencing reactions were then purified and precipitated using Ethanol/EDTA/Sodium acetate precipitation method. Capillary sequencing was performed in a Genetic Analyzer 3500 (Applied Biosystems, Thermo Fisher Scientific, USA) and the data were  analyzed using Sequencing Analysis software. The sequences were aligned with the published sequence of the <em>ILDR1</em> gene (NM_001199799.1).</p>

[STEPS]
TITLE: Protein Estimation by BCA
AUTHORS: Vidur Sabharwal

[STEPS]
?. Make the following dilutions of autoclaved dd. H20 in triplicatesBSA conc(µg/ml)      0            5            10            15              20            25Water(µl)                    25          22.5       20            17.5          15            12.5BSA(2mg/ml)(µl)      0            2.5          5              7.5            10            12.5
?. Dilute sample in the following dilution in triplicatesWater(µl)       23Sample(µl)    2
?. Total wells(x) = 18(controls) + 3*n(samples)Make final BCA solution for x+1 wells with 200µl per well and 1 part Reagent A and 50 parts Reagent B from The Pierce BCA assay kit.
?. Incubate at
65 °C
?. Take readings immediately after concentration estimation in a spectrophotometer at 562nm. Make sure the OD is not above 1. If so, do not analyze the data
?. Export the results in Excel, fit the standards to a linear line and calculate the samples concetration.The actual concentration of the sample will be O.D./2*slope(standards)
TITLE: Rotarod test
AUTHORS: michela.deleidi, Bianca Marchetti, Federico Bertoli, Carmela Giachino, carmela.giachino

[DESCRIPTION]
Rotarod test was used to evaluate the effect of LPS treatment on motor performance during aging process between genotypes.

[STEPS]
SECTION: ROTAROD
1. To test motor performance, we used the accelerated Rotarod (five-lane accelerating rotarod; Ugo Basile, Comerio, Italy), a motor behavioral test widely used to assess motor deficit in neurodegenerative disease models in rodents. Mice have to keep their balance on a horizontal rotating rod (diameter, 3 cm) and rotation speed is progressively increased every 30 sec by 4 rpm.


SECTION: ROTAROD
2. Before the test, WT and G2019S within the different treatment groups were housed five per cage and acclimated to a 12h shift in light/dark cycle so that the exercise occurred during the animal normal wake period.

SECTION: ROTAROD
4. The Rotarod performance was assessed at day -7, 0 days, and during NaCL/LPS injection paradigm, at monthly intervals, for the determination of motor deficit (Supplementary Figure 1).
SECTION: ROTAROD
3. To start the trials, the mice (five mice are tested at the same time) are placed on a rotating rod; when the mice fall down or when 5 min are completed, a switch is activated that automatically stops a timer. On the day of testing, the mice perform 5 trials, separated by an interval of 30 min between each trial.
TITLE: SPARC_Duke_PelotGrill_OT2-OD025340_RatVagusNerve_Morphology
AUTHORS: Nicole A. Pelot, Gabriel B. Goldhagen, Jake E. Cariello, Warren M. Grill

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The protocol describes image segmentation and image analysis methods to quantify rat vagus nerve morphology from Masson's trichrome-stained nerve cross sections.</div></div>

[STEPS]
?. [Image Segmentation]
We used Nikon's NIS Elements software (v5.02.01, Build 1270) to segment rat vagus nerve micrographs stained with Masson's trichrome using the General Analysis RGB tool.
?. [Image Segmentation]
We converted the binary segmented image into “Graticule Masks”, binary images saved as TIFs.
?. [Image Analysis]
We imported the TIFs into Matlab and generated a data structure of the x and y coordinates of the pixels for each closed boundary of the loaded binary images using the bwboundaries function.
?. [Image Analysis]
We stored the pixel coordinates with indexing that assigns a fascicle number, which were then checked so that both the interior and exterior perineurium trace relate to the same fascicle.
?. [Image Analysis]
We scaled the pixel coordinates to microns using the segmented scale bar.
?. [Image Segmentation]
For each image, we selected preprocessing steps, such as smoothing and sharpening.
?. [Image Segmentation]
For each image, we selected ranges of hues, saturations, and intensities to values that identify the perineurium and, in multifascicular cases, different values to identify the entirety of the nerve. For the cervical samples, we identified the vagus as the largest nerve in the carotid sheath. For the subdiaphragmatic samples, we identified and segmented the largest fascicle on the anterior surface of the esophagus; we also segmented smaller fascicles nearby if they were approximately within double the diameter of the largest fascicle in distance from the edge of the largest fascicle. For monofascicular nerves, the outer perineurial boundary was used as the nerve boundary. For multifascicular nerves, the nerve trace was identified as the approximate transition from denser connective tissue grouping the fascicles to looser connective tissue.
?. [Image Segmentation]
For each image, we selected postprocessing steps, such as setting a minimum size criterion (eliminate small off-target regions), smoothing, cleaning, closing, and filling holes.
?. [Image Segmentation]
We made manual adjustments as needed, including manual deletion of off-target regions and filling of target areas that had not been captured.
?. [Image Analysis]
We calculated cross-sectional area of each fascicle (inner perineurium and outer perineurium traces) and nerve using Matlab’s polyarea. Effective diameter (for a nerve or fascicle) is the diameter of the circle that has the same cross-sectional area as the raw trace. The perineurium thickness is half of the difference in effective diameters of the inner and outer perineurium traces.
TITLE: Single-limb fast FR4 operant task (Mendonça et al 2024)
AUTHORS: Marcelo D Mendonça

[DESCRIPTION]
Single-limb fast FR4 operant task methods from Mendonça et al 2024.

[BEFORE_START]
Mice were placed on food restriction throughout training, and fed daily after the training sessions with approximately 1.5 -2.5g of regular food to allow them to maintain a body weight of around 85% of their baseline weight. 

[GUIDELINES]
A behavioral experiment control system built around the Micropython microcontroller, was used to control and detect events and supply rewards. Licks were detected using an infrared beam and through a side camera, mouse position in the box was monitored through a camera placed on the top of the box.

[STEPS]
SECTION: Single-limb fast FR4 operant task behavior
1. At the beginning of each session there was the onset of a light, and the animals were required to perform a sequence of presses at a minimum frequency in order to obtain a sucrose reward. 
SECTION: Single-limb fast FR4 operant task behavior
2. Session 1: To facilitate learning, animal are initially exposed to one session of magazine training where sucrose would be available on a random time schedule. 
SECTION: Single-limb fast FR4 operant task behavior
3. There are then 3-4 sessions of continuous reinforcement schedule (CRF) before training, where single lever presses would be reinforced.
SECTION: Single-limb fast FR4 operant task behavior
4. In the following sessions animals were reinforced if they performed a sequence of 4 consecutive presses (Fixed Ratio 4, FR4) in a particular time window (FR4/Xs, fixed-ration four within X seconds). 
SECTION: Single-limb fast FR4 operant task behavior
4.1. The duration of time required to perform the four lever presses was reduced across sessions from 100 seconds to 20 s, 8 s, 4 s, 2 s and finally 1s. To shape animals to use only one of the forelimbs the lever was progressively retracted and the slit thought which the forelimb accessed the lever was reduced with a custom-built piece. 
SECTION: Single-limb fast FR4 operant task (Imaging group)
5. Animals perform the task 2 times/session - one with the lever in the left side of the box and the other with the lever in the right - that were randomized throughout the training. Task ended after 30 minutes on each side or when the animals obtained 30 rewards. 
SECTION: Single-limb fast FR4 operant task (Imaging group)
5.1. The lever was equipped with a digital 9-axis inertial sensor with a sampling rate of 200 Hz (MPU-9150, Invensense) assembled on a custom-made PCB and connected to a computer via a custom-made USB interface PCB (Champalimaud Foundation Hardware Platform). Lever velocity was extracted from this sensor. Timestamps from the behavioral task were synchronized with calcium imaging data using TTL pulses sent from the behavioral chambers to the Inscopix data acquisition system via a BNC cable.
TITLE: GCaMP6f imaging using a mini-epifluorescence microscope (Mendonça et al 2024)
AUTHORS: Marcelo D Mendonça

[DESCRIPTION]
GCaMP6f imaging using a mini-epifluorescence microscope methods from Mendonça et al 2024. 

[BEFORE_START]
Mice are briefly anesthetized using a mixture of isoflurane and oxygen (1% isoflurane at 1L/min) and the mini-epifluorescence microscope was attached to the baseplate. 

[GUIDELINES]
This procedure excludes surgical procedures that would occur prior to the imaging day. Please see 'virus injections and lens placement procedure' for those details. 

Fluorescence images were acquired at 10 Hz and the LED power was set 40-60% with a gain of 4

[STEPS]
SECTION: GCaMP6f imaging
1. Once the baseplate (that is attached to the mouse) is attached to the mini-epifluorescence microscope, mice are recovered in their home cage for 15-20 minutes prior to starting imaging. 
SECTION: GCaMP6f imaging
2. With the baseplate attached to the microscope, mice are them imaged during the FR4/1s task in 3 performance days. (see Single-limb fast FR4 operant task methods for details)
SECTION: GCaMP6f imaging
1.1. Image acquisition parameters were always set to the same parameters between sessions to be able to compare the activity recorded.
TITLE: Prepare 2L of 2X freezer buffer to freeze down C. elegans
AUTHORS: Cancer Research UK / Wellcome Gurdon Institute media kitchen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol accompanies you in the preparation of 2 litres of 2X freezer buffer, which is the buffer used to freeze down </span><span style = "font-style:italic;">C. elegans</span><span> strains for long-term storage</span></div></div>

[STEPS]
?. 
?. ABC1IngredientsQuantity2NaCl&nbsp; 1M200ml31M KH2PO4&nbsp;pH 6100ml4Glycerol 600ml5Double
  distilled H2Oup to 2L6  Autoclave7   80.1M
  MgSO4&nbsp;300 microlitres9  per 100ml bottle
ABC1IngredientsQuantity2NaCl&nbsp; 1M200ml31M KH2PO4&nbsp;pH 6100ml4Glycerol 600ml5Double
  distilled H2Oup to 2L6  Autoclave7   80.1M
  MgSO4&nbsp;300 microlitres9  per 100ml bottle
?. AB11Combine NaCl, KH2PO4 and Glycerol solutions with approx 900ml double distilled H2O in 2L measuring
  cylinder on magnetic stirrer.22Make up to volume using double distilled H2O33Aliquot into 20x100ml media bottles,label,date and autoclave.44After autoclaving and the solution is cold,add 300 microlitres
  of 0.1m MgSO4 to
  each 100ml bottle using a Gilson in the flow hood.
AB11Combine NaCl, KH2PO4 and Glycerol solutions with approx 900ml double distilled H2O in 2L measuring
  cylinder on magnetic stirrer.22Make up to volume using double distilled H2O33Aliquot into 20x100ml media bottles,label,date and autoclave.44After autoclaving and the solution is cold,add 300 microlitres
  of 0.1m MgSO4 to
  each 100ml bottle using a Gilson in the flow hood.
TITLE: Marble Burying 
AUTHORS: Tim Sampson

[DESCRIPTION]
Description: Marble burying is a typical behavior in mice that has
been shown to be sensitive to differences based on strain, hippocampal lesions,
and biological/chemical treatments1,2,3,4. The marble burying assay
is quick and requires no specialized equipment to perform. Marble burying
relies on the assessment of naturalistic behaviors that have been attributed to
anxiety-like or OCD-like movements5. In this assay experimenters will allow mice to freely bury marbles placed on top of bedding, mice will be given a determined amount of time to bury these marbles (30 minutes used by our lab) and the number of marbles remaining uncovered will be quantified and compared between genotypes, treatment groups, ages. Overall, the lack of marble burying is indicative of alterations to natural instincts of mice, suggesting perturbations to neurological systems.

[STEPS]
SECTION: Set-up
1. The night before assessment gather and autoclave enough standard mouse cages for each mouse to be assessed.
SECTION: Set-up
3. Day of assessment fill each cage with 1.5" of sterile unscented mouse bedding.
SECTION: Set-up
2. Autoclave enough marbles to have 20 per cage (marbles should be flat on one side and rounded on the other, see materials tab for reference).
SECTION: Set-up
4. Arrange the marbles in each cage so that the flat edge is contacting the bedding and rounded edge exposed. The arrangement should be uniform in each cage and follow what is seen in Figure 1).
 

SECTION: Set-up
5. Place each cage on the bench of the behavior room (or any space free of noise and commotion) with a numeric label associated with each cage (1-n).
SECTION: Acclimation
6. Once all cages are set up, bring mice up from the vivarium to acclimate (at least 1h)
SECTION: Acclimation
7. Determine the order of the mice for each cage, making note of this order in your lab notebook.
SECTION: Marble Burying
8. After acclimation, start to place the mice 1-by-1 into the labeled cages. Mice should be placed into the corner of each cage (remain consistent), making sure to gently place the mouse into the bedding to avoid from disturbing the marbles.
SECTION: Marble Burying
9. As soon as the first mouse is in the cage, start a timer for 30 minutes and continue to place the rest of the mice into their cages.
SECTION: Marble Burying
10. After 30 minutes is up, remove mice from their cages in the same order they were placed in. Again, do so cautiously to avoid from disturbing the bedding or marbles at this step.
SECTION: Marble Burying
11. Once all mice are removed, take a photo of each cage, with the respective cage number visible.
SECTION: Marble Burying
12. Send images to 2-3 independent reviewers who are blinded to the order of mice.
SECTION: Scoring
13. Have each reviewer record the number of "non-buried" marbles in each cage. "Non-buried" marbles are those that are >25% visible. 
SECTION: Scoring
14. Average the amount of marbles buried between mice of the same genotype, treatment, age, etc. and compare between groups.
TITLE: SDS-PAGE
AUTHORS: Ana Belem García González, Georgina Diego, Irán Alessandra Chaparro Rodríguez, Jair Alexis Gardea Sáenz

[DESCRIPTION]
This protocol shows the steps carried out by team Tec-Chihuahua to perform SDS-PAGE

[STEPS]
SECTION: Protocol
1. Clean the components of the electrophoresis camera with 70% ethanol and gauze 's. 
SECTION: Protocol
2. Assemble the chamber and check that there are no leaks by pouring distilled water between the glasses.
SECTION: Protocol
3. Prepare polyacrylamide gels:


 

SECTION: Protocol
4.  Pour the solution between the glasses with a 1 mL micropipette, leaving a space of 1.5 cm for the concentrating gel. To level, distilled water is added and allowed to settle for 30 min or until a line is seen between the gel and the water.

 

SECTION: Protocol
5. Pour solution onto separating gel using a 1 mL micropipette.
SECTION: Protocol
6. 30 min 30 min Insert the comb (carefully avoiding the formation of bubbles) and leave to solidify for 30 min

SECTION: Protocol
7. When the gels are polymerized, prepare an electrophoresis chamber with 1X running buffer until it covers the gels and it reaches the line of two gels.
SECTION: Protocol
8. Sample PTake the pellets contained in Eppendorf tubes 
Take the pellets contained in Eppendorf tubes
Add 300 µL of 1X loading buffer and resuspend the pellet.reparation:

SECTION: Protocol
9. To denature proteins, heat samples in boiling water for 5 min.
SECTION: Protocol
10. Load gels with the hot sample.
SECTION: Protocol
11. In the first well add 7 µLof the molecular weight marker.

SECTION: Protocol
12. Once the samples are loaded, run the gel at 80 V for 20 minand then at 180 V for 45 min

SECTION: Protocol
13. Turn off the camera and disarm it.
SECTION: Protocol
14. Remove the gels from the glasses and place in a container with staining solution. Leave stirring for one hour.
SECTION: Protocol
15. Remove staining solution after one hour, add destaining solution and leave stirring for 20 min Change the destaining solution and leave again for 20 min

SECTION: Protocol
16. Leave stirring until the gel is transparent.
SECTION: Protocol
17. Analyze the gels.
TITLE: TPGY BROTH (TRYPTONE PEPTONE GLUCOSE YEAST EXTRACT BROTH)
AUTHORS: Roey Angel, Ana Lara-Rodriguez, Eva Petrova

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">For the enrichment of Clostridium sp. from soil samples, sufficient for 100 cultivation tubes.</div></div>

[STEPS]
?. [Medium preparation]
Final pH ( at 25°C) 7.0±0.2
[Casein Hydrolysate]
[Peptic Digest from Animal Tissue (Peptone A)]
[Yeast Extract]
[Dextrose]
[Sodium Thioglycolate ]
Heat if necessary, to dissolve the medium completely.
?. [Medium preparation]
Sterilize by autoclaving at 15 lbs pressure  for  .
121 °C
?. [Medium preparation]
Refrigerate  the sterile medium until use.
4 °C
?. [Before inoculation of media]
Pour   of media in sterile tubes.
10 mL
?. [Before inoculation of media]
Heat media in water bath   OR alternative do a gass exchange using the gassing station for 3 minutes to each tube OR put tubes in to the anaerobic tent for   to  before inoculating.
100 °C
TITLE: Bias in Bias Recognition - Research Materials
AUTHORS: Qi Wang, Hee Jin Jeon

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Biases perpetuate when people think that they are innocent whereas others are guilty of biases. We examined whether people would detect biased thinking and behavior in others but not themselves as influenced by preexisting beliefs (myside bias) and social stigmas (social biases). The results of three large studies showed that, across demographic groups, participants attributed more biases to others than to themselves, and that this self-other asymmetry was particularly salient among those who hold strong beliefs about the existence of biases (Study 1 and Study 2). The self-other asymmetry in bias recognition dissipated when participants made simultaneous predictions about others’ and their own thoughts and behaviors (Study 3). People thus exhibit bias in bias recognition, and this metacognitive bias may be remedied when it is highlighted to people that we are all susceptible to biasing influences. </div></div>

[STEPS]
?. [Bias in bias recognition]
TITLE: Gene expression analysis by quantitative Real-Time PCR (qPCR)
AUTHORS: miquel.vila

[DESCRIPTION]
Quantitative Real-Time PCR (qPCR)

[STEPS]
SECTION: Thermocycling
3. Perform PCR using the following cycling conditions in a LightCycler® 480 System (Roche):	
50˚C for 2min
95˚C for 2min
95˚C for 15s + 60˚C for 1min (x40cycles)
SECTION: Data an
4. Using the LightCycler Software obtain the threshold cycles (CT) signals from each samples.
SECTION: Data an
5. Perform ΔΔCT-method to analyze the expression data using the endogenous control genes and the reference experimental group.
SECTION: qPCR plate reagents
1. Option 1: TaqMan qPCR
SECTION: qPCR plate reagents
1.1. Prepare Taqman Master mix (ul per well):
5ul: Taqman Gene Expression Master Mix (Applied Biosystems, # 4369016)
0.5ul Taqman Gene Expression Assays 

SECTION: qPCR plate reagents
1.3. Add 4.5ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics)
SECTION: qPCR plate reagents
2. Option 2: SYBR qPCR
SECTION: qPCR plate reagents
2.1. Prepare SYBR Master mix (ul per well):
5ul PowerUp SYBR Green Master Mix (#A25776, Applied Biosystem-ThermoFisher) 
1ul Primers (Forward and Reverse, diluted in Nuclease-free water at 5µM)

SECTION: qPCR plate reagents
2.3. Add 4ul of cDNA per well (10ng cDNA) in technical triplicates to each well of LightCycler 480 Multiwell 384-plate (#4729749001, Roche Diagnostics)
SECTION: qPCR plate reagents
2.2. Add 6ul of SYBR Master mix to each well with an automatic pipette (Multipette E3, Eppendorf)
SECTION: qPCR plate reagents
1.2. Add 5.5ul of Taqman Master mix to each well with an automatic pipette (Multipette E3, Eppendorf)