eamag/protocols-with-steps huggingface dataset:
TITLE:   ELISA for quantification of IL-3 in human serum.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Interleukins (IL) are a type of cytokine first thought to be expressed by leukocytes alone but have later been found to be produced by many other body cells. They play essential roles in the activation and differentiation of immune cells, as well as proliferation, maturation, migration, and adhesion. They also have pro-inflammatory and anti-inflammatory properties. The primary function of interleukins is, therefore, to modulate growth, differentiation, and activation during inflammatory and immune responses. Interleukins consist of a large group of proteins that can elicit many reactions in cells and tissues by binding to high-affinity receptors in cell surfaces. They have both paracrine and autocrine function. Interleukins are also used in animal studies to investigate aspect related to clinical medicine.T cells and stem cells make IL-3. It functions as a multilineage colony-stimulating factor </span><a href="https://www.ncbi.nlm.nih.gov/books/NBK499840" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">[1]</span></a></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Justiz Vaillant AA, Qurie A. Interleukin. In:</span><span style = "font-style:italic;">StatPearls</span><span>. Treasure Island (FL): StatPearls Publishing; June 12, 2019.</span></div></div>

[STEPS]
?. An anti-human IL-3 coating antibody is adsorbed onto microwells by incubation overnight at 4°C.
?. Add 50 µl  of human serum. Human IL-3 present in the serum sample binds to antibodies adsorbed to the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wasth o remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-human IL-3 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and binds to the biotin-conjugated anti-human IL-3 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) to each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of human IL-3 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IL-3 standard dilutions and the human IL-3 sample concentration determined.
?. For better results place the microplate on a plate shaker in every incubation.
TITLE: JAX - EZ Lysis Nuclei Isolation for 10x Genomics Assays
AUTHORS: Shannon Bessonett, Greggory  A Perry, Sandra L Daigle, Paul.gabriel  , Diane  Luo, Jessica  Grassmann, William F F Flynn, Elise  Courtois, Paul  Robson

[DESCRIPTION]
The purpose of this protocol is to produce single nuclei from frozen human tissues for downstream assaying with the  10x Genomics Multiome assay.  

This protocol has been demonstrated using Human placenta tissue as well as human Glioblastoma.

This protocol is modified from Sigma Aldrich Nuclei Isolation Kit: EZ Prep protocol and 10x Chromium Demonstrated protocol for Single Cell Multiome ATAC + Gene Expression Sequencing (CG000365).

[BEFORE_START]
Familiarize yourself with 10x Genomics protocol CG000365.

[GUIDELINES]
Assess nuclear membrane integrity (refer to 10x Genomics CG000365), using Brightfield or Trypan blue
Assess for debris, if large amount of debris present, filter with smaller pore size to decrease debris. 
Use either Trypan blue and Countess (for clean average size nuclei preps) or AO/PI and Nexcellom K2 counter (if nuclei prep has debris)
If RNA content is of concern Imaging nuclei can be done with fluorescence, and Syto RNA stain


[STEPS]
SECTION: Sample Prep
1. Slightly thaw sample out on ice and place onto pre chilled Petri dish on ice.
SECTION: Sample Prep
2. Cut tissue into small pieces and add to a microcentrifuge tube.
SECTION: Cell lysis
3. In micro centrifuge tube containing the tissue add 100 µL of lysis buffer.

SECTION: Cell lysis
4. Using a plastic pestle to grind tissue by pushing pestle down and twisting to break up the tissue. 
Start timer once lysis buffer is added.
SECTION: Cell lysis
5. Add 25 µL to wash off pestle and place tube on ice for a total of 5 min.

SECTION: Cell lysis
6. After 5 minutes of lysis on ice, 500 x g, 5 min, 4 °C.

SECTION: Cell lysis
7. Remove supernatant and resuspend pellet using wide bore tips in 100 µL.

SECTION: Cell lysis
8. Incubate on ice for a total of 5 min.

SECTION: Washing
9. Add 500 µL and 500 x g, 5 min, 4 °C.

SECTION: Washing
10.   
SECTION: Washing
11. After last wash, filter suspension through 40um filter.
SECTION: Washing
12. Resuspend sample in 1x Nuclei buffer (10x provided) containing RNase inhibitor, and DTT.
SECTION: Load
13. Load for transposition and continue with the 10x multiome protocol.
TITLE: FCMPASS - Creating a cytometer database and datasets
AUTHORS: Joshua Welsh, Jennifer Jones

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol outlines the steps required to create flow cytometer databases and datasets using the FCMPASS software. This is one of a number of protocols in the pipeline for performing small particle calibration using the fcmpass software package.</div></div>

[STEPS]
?. Open FCMPASS.
?. Click the ‘+’ icon next to ‘Cytometer IDs’ list and enter a unique name to identify a instrument.
?. Select the relevant cytometer ID to add the dataset to
?. Click the ‘+’ icon next to the ‘Datasets’ list.
?. In the window enter the acquisition date of the calibration data and the dataset/experiment name. If there are any notes related to the experiment that are beneficial, they can be entered in the ‘Dataset Notes’ field.
TITLE: S1: Step-by-step-guide using image J in the work flow.
AUTHORS: Paul Kalke, Conrad  Helm

[DESCRIPTION]
How to use ImageJ in our 3D-workflow from data manipulation, import, segmentation and export as OBJ-file.

[STEPS]
2.   
•Make sure your Image Sequence starts with 1 and choose Use virtual stack to save RAM
•You can scale your data set down too 

1.   
•Open Image J and import your Image Sequence
•It is possible in Image J to transform stacks into Images Sequences and vice versa
5.   
•Import your binned data set by right click in blank (black window) and select folder of binned data set
6.   
•Choose slice thickness depending on your data set, adjustments are possible in all programs
7.   
•To create different labels, each for a structure you want to reconstruct, you need a new child and give it the name of your label
3.   
•Make sure your data set is processablefor your computer
•Binning is easy and suitable for reducing data size, here bin2 from 14.3 to 3.4 GB
•Only bin x and y not z
•Save binned data set
4.   
•For segmentation, useTrakEM2
•Open blank
8.   
•Then right click on the new child and create an area_list
9.   
•Drag `n`drop the named folder (child) together with the area_list
•Right click on the area_list and add a new area_list
10.   
•Name the new area_list like the child in the beginning
•save your Trak EM2 file by press save in main tool bar (.xml-file)
11.   
•Now your named label is available forsegmentation under z space
12.   
•Now start to segment your structure
•Make sure the brush tool in the main tool bar is checked
•Hot keys and how to reconstruct:
-Mouse wheel (mw); , and . : scrolling through data set
-Ctrl + mw; + and - : zoom in and out
-Shift + mw: size of pen
-Left mouse button (l mb): mark
-Shift + l mb: fill
-Alt + l mb: erase
-Alt + shift + l mb: delete all on slice
-Right mouse button (r mb) – Area: for all kind of interpolations
13.   
•after finishing your segmentation it is time to export your labels
•it is possible to export different labels solely, or together, but then they work like one
•e.g. in this example it is smart to export all parts of the cuticle as one label
14.   
•hide all labels you do not want to export in this turn by unchecking the eye button in the Z space
15.   
•now turn the "background" black, go to patches and push the fader on top to the left
•now you should only see your marked label, check the whole stack
16.   
•open the 3D viewer
17.   
•you can choose in Display as between surface or volume
•for later work with MeshLlaband Blender it doesn´t matter
•if you are not familiar with ImageJ keep the other parameters as in the shown image
18.   
•press ok and wait for the result
•if it is stretched or clinched don´t bother, you can change later or go back to the TrakEM2 window (see last step)
•save label as wavefront-file (.OBJ)
19.   
•use parameters as shown in image, press ok
20.   
•wait for the little black dot in the main tool bar before you export other labels
21.   
•if you want to change the slice thickness go to Layers of the TrackEM2 window, select all slices and right click
•Choose a factor for all slices
TITLE: Immunohistochemistry of porcine enteric neurons
AUTHORS: Gemma   Mazzuoli-Weber, Michael Schemann, Kristin Elfers, Birgit Kuch, Susanne Hoppe

[DESCRIPTION]
This protocol is to investigate the presence and localization of neurochemical markers in the submucosal and myenteric plexus of the porcine colon using immunhistochemical staining techniques. 

[STEPS]
1. Samples of colon were taken from apparently healthy pigs, placed in ice-cold oxygenated Krebs solution for preparation and immediately transferred to the laboratory. Tissues were then dissected in the ice-cold oxygenated Krebs solution for preparation to obtain whole-mount inner submucosal plexus or myenteric plexus preparations.
3. The preparations were then incubated in PBS/NaN33/horse serum for 1h at room temperature followed by 12h and 2h incubation with the primary and secondary antibodies, respectively.
4. Specimens were washed in PBS, mounted on object slides and cover slipped with a solution of PBS (pH 7.0)/NaN3 containing 65% glycerol.
5. The preparations were examined with an epifluorescence microscope equipped with appropriate filter blocks. Pictures were acquired with a camera connected to a computer and controlled by Olympus cellSens Standard Software. 
2. Tissue specimens were fixed overnight in a solution containing 4% paraformaldehyde and 0.002% picric acid in 0.1 mol/l phosphate buffer and then washed (3 x 10 min) in PBS.
6. Pictures of the stained ganglia were acquired.
TITLE: Gravimetric Water Content (GWC)
AUTHORS: maggie.bowman

[DESCRIPTION]
The method is the mass of water per mass of dry soil. 

[STEPS]
1. Record the mass of the empty labeled tin in the Core processing file. Add 10 g and record the mass of the tin and soil (this should be completed in the core processing step. 

2. Place the soils in the drying oven for 1440 min at100 °C.

3. After 1440 min remove samples from drying oven and allow to cool. Record the mass of the soil and tin. 

4. Return samples to the drying oven for an additional 1440 min. 

5. Repeat step 3 and record the new mass. If the mass is still changing repeat steps 3 and 4 until no further change.
1.1. Do not tare the tin.
TITLE: Buck Institute Morphology H &amp; E staining protocol
AUTHORS: sbreslin

[DESCRIPTION]
 Histological Stain for FFPE slides
RESULTS:  Nuclei-blue; Cytoplasm, red blood cells and connective tissue-shades of pink

[STEPS]
SECTION: Hematoxylin and Eosin Staining
1. Deparaffinization and rehydration: 2 x xylene 7 min, 100% ethanol 4 min, 95% 4 min, 80% 4 min, 70% 4 min, bring to water.
SECTION: Hematoxylin and Eosin Staining
2. Hematoxylin staining for 30 seconds - 4 min. using filtered dye (usually 2 mins) - Modified Mayes’s Hematoxylin HXMMHPT StatLab Medical Products
SECTION: Hematoxylin and Eosin Staining
3. Rinse in ddH20 x2
SECTION: Hematoxylin and Eosin Staining
4. 10% acetic acid rinse (acid into water) – 1 min
SECTION: Hematoxylin and Eosin Staining
5. rinse in ddH20 x2
SECTION: Hematoxylin and Eosin Staining
6. TBS (1x) rinse or Scott’s Tap Water (1L + 3.5g Sodium Bicarb + 20g Magnesium Sulfate pH 8.0)  - 2 mins
SECTION: Hematoxylin and Eosin Staining
7. Rinse in ddH20
SECTION: Hematoxylin and Eosin Staining
8. Rinse in 70% ETOH
SECTION: Hematoxylin and Eosin Staining
9. Stain Eosin for 0.5 to 2 minutes (keep an eye on intensity of pink) – Eosin Y Stain STE0150 StatLab Medical Products
SECTION: Hematoxylin and Eosin Staining
10. Rinse in 90% EtOH
SECTION: Hematoxylin and Eosin Staining
11.  Rinse in 95% EtOH, 100% EtOH,  xylene
SECTION: Hematoxylin and Eosin Staining
12. Coverslip with Permount Mounting Medium EMS #17986-01
TITLE: Nucleoside analysis with high performance liquid chromatography (HPLC) 
AUTHORS: Atanas Radkov

[DESCRIPTION]
This protocol details the detection of modified nucleosides using HPLC. 

[STEPS]
SECTION: Run samples
5. Begin the gradient at 100% solution A, then ramp up to 25% solution B over 16 min, then ramp down to 0% solution B in 1 min, and finally stay at 0% solution B for 13 min (30 min total method time). Flow rate for the entire run should be 0.5 mL per min. Detect elution of compounds at 260 nm.
SECTION: Run samples
7. Make sure the HPLC column is re-equilibrated in solution A, then inject 10 µL of your experimental sample. Run the same program used for the standards. Perform in triplicate to obtain an average retention time and a standard deviation.
SECTION: Run samples
4. Equilibrate the column with solution A. Once fully equilibrated, inject 10 µL of one of the standards.
 

SECTION: Analysis
8. Extract peak elution times from the HPLC software report. Common calculations can include calculating average elution time and standard deviation, if you have ran replicates. We also often plot the A260 signal to look at the shape of individual peaks. 
SECTION: Run samples
6. Run in triplicate to obtain an average retention time and a standard deviation. Repeat for each standard.
SECTION: Prepare samples and solutions
1. Prepare the following standards at 100 µM by dissolving each in a small volume of HPLC-grade methanol and bringing up the volume as necessary with MQ water. Filter each solution through a 0.22 µm syringe filter (we used SLGSV255F - Millipore-Sigma), and keep solutions at 4 °C prior to HPLC analysis.
 

SECTION: Prepare samples and solutions
2. Prior to HPLC analysis, adjust the total experimental nucleoside sample volume to 100 µL by diluting with MQ water and then centrifuge each sample at 21000 × g for 15 min to remove any debris.


 

SECTION: Prepare samples and solutions
3. Prepare solutions A and B. Filter solution A through a 0.22 µm filter (we used 10040-440 VWR) and then adjust pH with 1 M HCl.
 

SECTION: Prepare samples and solutions
3. Prepare solution A. Filter solution A through a 0.22 µm filter (we used 10040-440 VWR) and then adjust pH with 1 M HCl.
 

SECTION: Run samples
4. Equilibrate the column with solution A. Once fully equilibrated, inject 10 µL of one of the standards.
 

SECTION: Run samples
5. Because this is an isocratic gradient, the method is just maintaining 100% solution A throughout the run. Flow rate for the entire run should be 0.5 mL per min. Detect elution of compounds at 260 nm.
SECTION: Run samples
6. Run in triplicate to obtain an average retention time and a standard deviation. Repeat for each standard.
SECTION: Run samples
7. Inject 10 µL of your experimental sample. Run the same program used for the standards. Perform in triplicate to obtain an average retention time and a standard deviation.
SECTION: Analysis
8. Extract peak elution times from HPLC software report. Common calculations can include calculating average elution time and standard deviation, if you have ran replicates. We also often plot the A260 signal to look at the shape of individual peaks.
TITLE: Adhesive Removal Test to assess sensorimotor deficits in parkinsonian mice
AUTHORS: natalia.lopezgonzalezdelrey, Zachary Gaertner, Elizabeth Phelan

[DESCRIPTION]
This behavior is used to asses fine motor movements in a mouse parkinsonian model. It checks for correct paw and mouth sensitivity (time-to-contact) and correct dexterity (time-to-remove).

[STEPS]
SECTION: Adhesive Removal Test
1. Total duration: 4 days
SECTION: Protocol
2. Place the animal cage in the testing room to allow the animals to acclimate. 60 min 

SECTION: Protocol
3. Acclimating mice to testing box. 1 min 

4. Apply the two adhesive tape strips with equal pressure on each animal paw so that they cover the hairless part of the forepaws. Place the adhesive in alternate order (right or left) each day and randomize it within each group. 3 min 

5. Video record the animal’s behavior in the testing cage. 2 min 

SECTION: Analysis
9. Different analysis can be made:
- Mean Time to Notice (T1,T2)
- Mean Time to Remove (T3,T4 - T1,T2)
Compare Mean Time to Notice vs Mean Time to Remove

Tip: in the post-analysis: you can distinguish between right and left paw for comparisons. This helps to detect limb differences.
Example:
- Mean Time to Notice Right (T1)
- Mean Time to remove Right (T3)
- Mean Time to Notice Left (T2)
- Mean Time to remove Left (T4)
6. The contact time is defined as the point that the mouse reacts to the presence of the adhesive tape strips. For each adhesive, the mouse may start by either shaking its paws and/or directly bring its paws to its mouth. Both behaviours indicate that the mouse has felt the adhesive tape and represent the end of the time to contact. Time 1: when the mouse has felt the first piece of tape. Time 2: when the second piece of tape is felt.
7. Continue to observe the animal’s behavior in the testing cage. The mouse may use its mouth, and to a lesser extent the other forelimb, to attempt to remove the adhesives. Time 3: when the first piece of tape is removed. Time 4: when the second piece of tape is removed. Distinguish between right and left paw for comparisons.
8. Return the mouse to its cage. After 24 h, repeat the procedure.
TITLE: mRNA extraction and cDNA preparation
AUTHORS: Shiyi Wang

[DESCRIPTION]
mRNA extraction and cDNA preparation

[STEPS]
1. **Thaw and Resuspend Cells** - Thaw cells stored in TRIzol (15596026, Invitrogen). - Resuspend cells in 1 mL of TRIzol.
2. **Add Chloroform** - Add 200 μL of chloroform to the samples.
3. **Centrifuge Samples** - Centrifuge the samples at 12,000 g for 15 minutes at 4°C.
4. **Collect Aqueous Phase** - Carefully collect the aqueous phase.
5. **Precipitate RNA** - Add GlycoBlue Coprecipitant (AM9515, Invitrogen) and isopropanol to precipitate the RNA.
6. **Wash RNA Pellet** - Wash the RNA pellet with 75% ethanol.
7. **Dry and Resuspend RNA** - Air-dry the RNA pellet. - Resuspend the RNA in 40 μL of nuclease-free water.
8. **Isolate RNA** - Isolate the RNA using the Zymo Research RNA Clean & Concentrator-5 Kit (R1014, Zymo).
9. **Quantify RNA** - Quantify the RNA using the Qubit RNA HS Assay Kit (Q32852, Invitrogen).
10. **Equalize RNA Concentrations** - Dilute the RNA to equalize concentrations.
11. **Generate cDNA Libraries** - Use the qScript cDNA SuperMix (101414-102, VWR) for cDNA library preparation. - Follow this temperature profile: - 25°C for 5 minutes - 42°C for 30 minutes - 85°C for 5 minutes
12. **Dilute and Store cDNA** - Dilute the resulting cDNA threefold. - Store the cDNA at −80°C.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol presents a simple modification of the SMART-seq2 (Picelle et al, 2014) and SCRB-seq (Soumillon et al, 2014) protocols, with the goal of combining the sensitivity of SMART-Seq2 with the improved cost and throughput of 3' single cell protocols. Reverse transcription (RT) and PCR proceed as in SMART-Seq2, but using 3' barcoded oligos from SCRB-seq. While most barcoded methods (including SCRB-seq) pool cells together after RT, we perform PCR individually in each well, as this negates the need for an additional exonuclease and purification step, and therefore maintains the same molecular sensitivity as SMART-Seq2. However, we pool amplified cDNA from single wells immediately after PCR, and generate a single tagmentation-based 3' cDNA library. While this protocol therefore has advantages in both sensitivity and throughput, the absence of an exonuclease step prior to PCR disables the use of unique molecular identifiers for quantification.</p>

[STEPS]
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TITLE: LB Broth Agar Plates with Antibiotics
AUTHORS: Bailey  Clark

[DESCRIPTION]
The abstract will be added later.

[STEPS]
SECTION: Preparation For Autoclave
1. Add 125 mL of deionized water to a 250 mL Erlenmeyer Flask.

SECTION: Preparation For Autoclave
2. Add 3.125 g  of LB Broth powder (25 g  per 1 L  of water)  to the water in the Erlenmeyer Flask.


SECTION: Preparation For Autoclave
3. Add 1.875 g  of agar to the water ( 1.5% of 125 mL ) in the Erlenmeyer Flask. 

SECTION: Preparation For Autoclave
4. Stir on stirring plate for 5 min .

SECTION: Preparation For Autoclave
5. Cover the opening of the Erlenmeyer Flask with aluminum foil. 
SECTION: Preparation For Autoclave
6. Place a 10 mm strip of autoclave tape on the aluminum covering the opening of the flask. 
SECTION: Autoclaving
7. 

Autoclave. 
 



SECTION: After Autoclaving
8. After autoclaving, place the flask in the water bath at 60°C for 15 min . 
 


SECTION: After Autoclaving
9. Add 1 µL  of the antibiotic stock solution per mL of LB agar in the flask.



SECTION: After Autoclaving
10. Stir on the stirring plate for 1 min .

SECTION: After Autoclaving
11. Pipette 5 mL  of the LB agar mix into 20 60 mm petri dishes. 

SECTION: After Autoclaving
12. Leave the surface of the agar 1/4 uncovered in the laminar flow hood for 30 min .
TITLE: Gastrointestinal transit
AUTHORS: Connor Monahan

[DESCRIPTION]
This protocol details measuring gastrointestinal transit time in mice

[STEPS]
SECTION: Procedure
1. Prepare a sterile solution of carmine red (300 µl; 6%; Sigma-Aldrich, Cat #C1022; St Louis, MO) suspended in 0.5% methylcellulose (Sigma-Aldrich, Cat #M0512; St Louis, MO).
SECTION: Procedure
2. Administer 0.3 mL of carmine red solution by gavage through a 21-gauge round-tip feeding
needle.

SECTION: Procedure
2.1. The time at which gavage takes place is recorded as T0.
SECTION: Procedure
3. Place each mouse in separate cages without bedding.
SECTION: Procedure
4. 60 min after gavage, monitor fecal pellets at 10 min intervals for the presence of carmine red. Total GI transit time is considered as the interval between T0 and the time of first observance of carmine red in the stool.