eamag/protocols-with-steps huggingface dataset:
TITLE: Mitochondria purification
AUTHORS: wusj, schekman

[DESCRIPTION]
This protocol describes how to purify mitochondria from cell culture

[STEPS]
SECTION: Mitochondria purification
1. HEK293T cells were trypsinized (0.05% Trypsin 5 min ) and collected by centrifugation (500g 5 min). 

SECTION: Mitochondria purification
2. Cells were washed twice with NKM buffer (1 mM Tris HCl, pH7.3, 0.13 M NaCl, 5 mM KCl, and 7.5 mM MgCl2), and resuspended in six packed cell volumes of homogenization buffer (10 mM Tris pH 7.4, 10 mM KCl, and 0.15 mM MgCl2). 

SECTION: Mitochondria purification
3. Cells were homogenized by 10 passages through a 22G needle. 
SECTION: Mitochondria purification
4. Cell homogenates were mixed gently with the same volume of 2.3 M sucrose solution and centrifuged at 1200×g for 5 min 4 °C  to remove unbroken cells and large cell debris. 

SECTION: Mitochondria purification
5. The recovered supernatant fractions were centrifuged at 7000×g for 10 min 4 °C . 

SECTION: Mitochondria purification
6. Mitochondria enriched in the pellet fraction were resuspended in three packed cell volumes of Mitochondria Suspension Buffer (10 mM Tris, pH 7.3, 0.15 mM MgCl2, and 0.25 mM
sucrose).
TITLE: Copy of Fabrication of Microneedle Patches
AUTHORS: Stephania Konstantinidi, Hana Samet

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The goal is to create a microneedle. patch, which, by applying it on a leaf, will extract amplification-ready DNA.</div><div class = "text-block">All MN patches used for DNA extraction were fabricated using  polydimethylsiloxane molds. These molds were fabricated by laser  ablation, and the dimension of each mold is approximately 10 mm × 10 mm,  which has 15 × 15 arrays of microneedle conical cavities. The height of  each cavity is 800 μm, and the diameters of the tip and base are 10 and  300 μm, respectively.</div></div>

[STEPS]
?. [Mold preparation]
Clean the polydimethylsiloxane molds.
?. [Mold preparation]
Heat deionised water to 100 degrees Celsius   using a becher.
100 °C
?. [Mold preparation]
Put the mold(s) into the water and stir softly.
?. [Mold preparation]
Remove the molds and let them dry in a chemical hood for 1-2 hours.
?. [Microneedle Fabrication]
Fabricate the Microneedles
?. [Microneedle Fabrication]
Stick the molds on the bottom of an adequate recipient using double-sided tape.
?. [A solutionPVA SPVA SolutionP]
Add 4g of poly(vinyl alcohol) (30− 70 kDa, 10 wt %) powder and stir until transparent.
?. [A solutionPVA SPVA SolutionP]
Heat 28g of deionised water to 110 degrees Celsius  .
110 °C
?. [A solutionPVA SPVA SolutionP]
Prepare the PVA solution
?. [Microneedle Fabrication]
Add 1.5 mL of PVA solution on top of the mold.
?. [Microneedle Fabrication]
Centrifuge at 4000 rmp for 20 minutes at 40 degrees Celsius.
?. [Microneedle Fabrication]
Let the microneedles dry under a chemical hood overnight.
?. [Microneedle Fabrication]
Remove the microneedles from the molds.
?. [Use Microneedles !]
The microneedles are ready for use.
TITLE: Mass Spectrometry Identification of Samples Separated by Liquid Chromatography
AUTHORS: Caroline Green

[DESCRIPTION]
As one of the most powerful separation and analysis technologies in the field of chemistry, liquid chromatography (LC) has developed rapidly since the 1970s. Both in terms of fundamental chromatography theory and instrument performance, it has been greatly improved and improved. LC exhibits high separation efficiency and accurate quantification in the analysis of complex samples. 

[STEPS]
SECTION: Guide for Mass Spectrometry Identification of Samples Separated by Liquid Chromatography
1. Main Instruments and Equipment
SECTION: Guide for Mass Spectrometry Identification of Samples Separated by Liquid Chromatography
3. Main reagents*1
SECTION: Guide for Mass Spectrometry Identification of Samples Separated by Liquid Chromatography
2.  Material
SECTION: Experimental Methods
4. Sample purification. Wash the protein with acetone pre-cooled at -20°C to remove impurities.
SECTION: Experimental Methods
5. Trypsin digestion of the sample*2
SECTION: Experimental Methods
6. Liquid chromatography column flushing and equilibration
SECTION: More
7.
TITLE: Microscopy-based bead protein-protein interaction assay
AUTHORS: Justyna Sawa-Makarska

[DESCRIPTION]
This protocol describes how to perform microscopy-based bead protein-protein interaction assay with GST-labelled proteins as baits and fluorescently labelled proteins as preys. The protocol requires to have purified proteins and allows to monitor protein-protein interaction in an equilibrium state. The fluorescent signal can be quantified.

[STEPS]
SECTION: Interaction assay
4. Pipette the preys onto 384-well glass-bottom microplate (Greiner Bio-One). The minimal volume to cover the bottom of a well is 20 μl. The final concentration of the prey may vary depending on the strength of the interaction, usually between 0.1-1 μM. The number of proteins in the well can be adjusted to the experimental set-up. Importantly, different preys should have different flourophores attached for detection (e.g.prey1-eGFP and prey2-mCherry).
SECTION: Bait preparation
3. Spin down the beads (4000 rpm, 2 min, 4 °C) and remove the supernatant. Wash the beads twice with 200 μl of pull-down buffer and finally add 20 μl of pull-down buffer to have 1:1 beads:buffer ratio.
SECTION: Bait preparation
2. Mix the beads with a GST-labelled bait protein at the final concentration between 1 - 10 μM in a pulldown buffer in a 0.5 ml Eppendorf tube. Incubate for at 4 °C for 1 h gently rolling or rocking. 
SECTION: Bait preparation
1. Wash 20 μl of Glutathione Sepharose 4B (Cytiva) beads slurry with 200 μl distilled water and with 200 μl of pulldown buffer (e.g. 25mM TRIS-Cl pH 7.5, 150mM NaCl, 1mM DTT). 
SECTION: Interaction assay
5. Pipette 1 μl of bait-coated beads to each well. Incubate the plate for 30 min in the dark at room temperature. Do not apply rocking or mixing.
SECTION: Signal detection
6. Image the beads in the wells with a microscope configured to detect fluorescent signal. When imaging beads for further quantification it is recommended to use a confocal microscope and to adjust the focus to take the plane in the middle of the beads. As the size of the beads vary it may be necessary to acquire more than one image of one field of view. For a reference also collect bright field images.
SECTION: Signal quantification
7. For signal quantification, in ImageJ software draw eight lines across each bead that is in focus. Take the maximum brightness value along each the line that was taken (see attached pdf file). 
SECTION: Signal quantification
8. To correct for background fluorescence draw a small rectangle that covers an empty area (beads-free) of each picture. Take the average (not maximum) brightness of that area and subtract from the maximal fluorescence for each bead from that image.
SECTION: Signal quantification
9. Calculate an average of the corrected maximum intensities of all the beads to get the final value of the signal intensity of the replicate. Repeat the same experiment at least three times for statistical analysis.
TITLE: Tissue sampling from museum specimens of mitten crabs
AUTHORS: Christine Ewers

[DESCRIPTION]
Simple protocol for sampling tissues from mitten crabs housed in natural history collections. Mitten crabs are somewhat unique because they have hair on their chelae from which DNA can be extracted. However, the protocol could also be used for other crabs (I am providing a few possible tissue sampling sites).The tissues are intended to be used for DNA extraction and museomic / genomic studies. The protocol is aimed at limiting contamination and specimen destruction while allowing reasonable throughput. 

[STEPS]
1. Clean all surfaces with DNAaway
2. Flame forceps and scalpel
3. Open collection jar
4. Take out one specimen
5. Place in clean tray with scale (e.g. ruler). Take first picture of the collection metadata (the jar label) and then picture of the crab. This way you can trace back which crab you photographed
6. Chose the tissue extraction site: leg that had already fallen off (1), muscle tissue from leg (2), hair from the chela
6.1. If legs have already fallen off the crab and are at the bottom of the jar, place it or a part of it in a 1.5ml Eppendorf tube
6.2. Cut a small window (5x5mm) in the ventral side of a walking leg with the scalpel and remove muscle tissue using forceps. Place in 1.5ml Eppendorf tube
6.3. Remove hair from 1/4 of one chela with scalpel, use forceps to hold hair and place in 1.5ml Eppendorf tube
7. Top off tube with the ethanol from the collection jar. Label with collection code (abbreviation of the collection, e.g. ZMH) and consecutive number (e.g. 01, 02, 03). Add collection information in spreadsheet. Make sure to link sampling ID (e.g. ZMH01) and collection ID (e.g. Cr24103) in spreadsheet.
8. Proceed to protocol: pH and formaldehyde measurement
TITLE: Stereology-mediated cell count using StereoInvestigator
AUTHORS: miquel.vila

[DESCRIPTION]
Protocol for cell counting using StereoInvestigator software

[STEPS]
1. Set slide properly on the microscope and set new reference point
2. Click on Probes → Optical Fractionator Workflow → Start a new subject  →
3. Enter cut thickness (5 µm (cut with microtome, 20 µm or 30 µm (cut with cryostat))
4. Enter interval according to the thickness: 17 if 5 µm, 6 if 20 µm or 4 if µm) 
5. In Select Low Mag Lens, click 4X
5.1. Click on Next Step
6. Select the Contour of Interest(s)
7. In Select High Mag Lens select 100X
7.1. Click on Next Step
8. In Manually enter the average mounted thickness enter the corresponding one
8.1. Click on Next Step
9. In Define the Counting frame enter 50 x 50 µm
9.1. Click on Next Step
10. Enter 25 Percentage
10.1. Click on Display Changes
11. Enter 100x75 µm (if cut thickness is 5 µm) or 125x100 (if 20 µm or 30 µm)
11.1. Click on Display Changes
12. In Optical Disector Height enter 5 µm, 20 µm or 30 µm 
11.2. Click on Next Step
12.1. Click on Next Step
13. Select your first section
13.1. Click on Yes
14. Select the desired region
14.1. Click on start counting
15. Focus on the tissue
15.1. Click on OK
16. Select a number of markers according to the desired cell types 
 

17. Count every defined region from every set section 
17.1. Click on Next Step
17.2. Click on Display Probe Run List
18. Click on Section Name → Click on Counter/Marker Name → Select all sections belonging to the same region
19. Click on View Results
 

20. Click on the different markers to see the number of cells counted and the estimated cell number
20.1. Click on CE Scheaffer to view the area if needed
20.2. Repeat steps 17 through 20.1 for each region
TITLE: Measles whole-genome sequencing
AUTHORS: My VT Phan, Matthew Cotten

[DESCRIPTION]
This is an optimised protocol for PCR amplification of the Measles virus (MV) genome using specific primers designed for B3 genotype. The method generates cDNA amplicons suitable for whole genome sequencing using the MinION platform. The method covers the entire ca. 16,000 nt MV genome in 8 overlapping amplicons of about 2200 bp (see Figure below). Both the reverse transcription and PCR steps are performed in  two separate  reactions to avoid short overlapping PCR products. 
The method has worked successfully with MV directly from oral fluid samples and with tissue culture passaged material.  Primer sequences can be found at the following GitHub site: https://github.com/mlcotten13/Measles_primers
  


[STEPS]
SECTION: PCR amplification of measles virus genome
1. cDNA synthesis
 

SECTION: PCR amplification of measles virus genome
1.2. For each FPM reaction, add the following:
4 µL 
1 µL 
1 µL 
1 µL 
1 µL 

Then incubate the reactions using the following conditions:
42 °C  for 50 min 
70 °C for 10 min 

SECTION: PCR amplification of measles virus genome
1.1. For each sample, prepare 2 separate reactions called "FPM_A" and "FPM_B". For each reaction, add the following:
Primers: 2 µL 
Template: 10 µL 

Then incubate the reactions using the following conditions:
65 °C  for 5 min 
on ice for 1 min 

SECTION: PCR amplification of measles virus genome
2. PCR amplifications
 
For each of the FPM_A and FPM_B reactions, prepare the mastermix for PPM_A and PPM_B, respectively. 
The mastermix for each PPM reaction include:
7 µL 
5 µL 
0.5 µL 
2 µL 
0.5 µL 

Add 10 µL  from reactions FPM_A or FPM_B to each of the corresponding tube for PPM_A or PPM_B, respectively.

Set up the PCR cycling conditions:
98 °C for 30 s 
Cycles 35-40 times using the following:    
Denaturation at  98 °C  for 15 s 
Anneal at 55 °C  for 45 s 
Extension at 72 °C  for 3 min 
Polish at 72 °C for 10 min 
Then hold at 10 °C for indefinite
 
 


SECTION: PCR amplification of measles virus genome
3. Running the PCR amplification products on Agilent Tapestation or agarose gel electrophoresis to check for products at approximately 2 - 2.5 kb.
For samples with amplifications for both PPM_A and PPM_B reactions, pool PCR products of PPM_A and PPM_B. This pooled PCR product can be used directly for library preparation for sequencing on MinION using LSK109 kits. For Illumina sequencing, the pooled PCR products will need to be sheared for appropriate size. For Ion Torrent sequencing, please refer to the manufacturer's instructions for library preparation.
TITLE:        ELISA for quantification of interferon gamma (IFN-γ) in human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">IFN-γ is a key player in driving cellular immunity. It is capable of orchestrating numerous and vital protective functions to enhance immune responses in cancers and infectious diseases. It exhibits its immunomodulatory effects by enhancing antigen processing and presentation,, inducing an anti-viral state, increasing leukocyte trafficking, boosting the anti-microbial properties of immune cells and by affecting cellular proliferation and apoptosis. [1]</div><div class = "text-block">Reference</div><div class = "text-block">Kak G, Raza M, Tiwari BK. Interferon-gamma (IFN-γ): Exploring its implications in infectious diseases.Biomol Concepts. 2018;9(1):64-79. Published 2018 May 30. doi:10.1515/bmc-2018-0007</div></div>

[STEPS]
?. An anti-human IFN-γ coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human IFN-γ present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-IFN-γ 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 it binds to the biotin-conjugated anti-IFN-γ 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) into each well.
?. Incubate the microwells in the dark for 20 min.
?. A colored product is formed in proportion to the quantity of IFN-γ present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human IFN-γ standard dilutions and the human IFN-γ sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Artificial sea water  
AUTHORS: Simon Blanchoud

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Three alternative solutions for artificial seawater (ASW) have been tested successfully on our </span><span style = "font-style:italic;">Botrylloides</span><span> colonies. For routine work, we use commercial sea salts (CSS), for most analyses we use the Cold Spring Harbor Protocols (</span><a href="http://doi.org/10.1101/pdb.rec068270" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">CSPH</span></a><span>) and for very clean work the K-depleted Phosphate-buffered saline (K-PBS). While, CSPH and K-PBS can be prepared at 10X, CSS should be prepared at 2X directly.</span></div><div class = "text-block">Mix to dissolve and adjust pH as well as salinity to your local conditions.</div></div>

[STEPS]
?. [2X CSS]
sea salt in  H2O.
70 g
1 L
?. [2X CSS]
Mix to dissolve.
?. [2X CSS]
Filter at 10 µm
?. [10x CSHP]
To prepare 10X CSHP take NaCl262.9g4.5MKCl7.4g100mMCaCl29.9g90mMMgCl2 6H2O60.9g300mMMgSO4 7H2O39.4g160mMH2O1000ml
NaCl262.9g4.5MKCl7.4g100mMCaCl29.9g90mMMgCl2 6H2O60.9g300mMMgSO4 7H2O39.4g160mMH2O1000ml
?. [10X K-PBS]
To prepare 10x K-PBS take NaH2PO4 · H2O8.4 g0.06 MNa2HPO434.2 g0.24 MNaCl262.9 g4.5 MH2O1000ml
NaH2PO4 · H2O8.4 g0.06 MNa2HPO434.2 g0.24 MNaCl262.9 g4.5 MH2O1000ml
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Diet-induced obesity through administration of a western-type diet high in fat and sugar to mice can result in a higher weight of adipose and liver tissues. The increased adipose tissue weight can be due to hypertrophy (bigger cells) or hyperplasia (more cells) of adipocytes, or a combination of both. Hematoxylin and eosin (H&amp;E) staining of adipose tissue sections allows you to investigate the size of the individual adipocytes. A western-type diet can induce accumulation of fat (= steatosis) in the liver, which is nicely illustrated by an H&amp;E stain.</p>

[BEFORE_START]
<p>-Filter hemaluin solution using a round Whatmann paper when the solution is covered with a film.</p>

[GUIDELINES]
<p>-Make sure during the hydration (step 1-5) and dehydration (10-14) steps that you allow most of each solution to drip off, before you transfer your slides into the next solution. This is to prevent contamination of the following solutions. This is especially critical in the dehydratation steps as otherwise too much water is transfered to the jars containing xylene and too many water droplets will have entered your mounted slides.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunostaining of H&amp;E Stained Paraffin Sections of Fly Heads
AUTHORS: Mel Feany

[DESCRIPTION]
This protocol describes how to perform immunostaining on H&E stained paraffin sections of fly heads.

[STEPS]
1. Embed the fly heads in paraffin and stain with H&E for neuronal counting, per the following protocol:
dx.doi.org/10.17504/protocols.io.4r3l24on4g1y/v1

2. Microwave slides in 10 millimolar (mM) sodium citrate for 15 min. 
Cool 20 min .

Stock: 100 millimolar (mM)  sodium citrate,  pH 6.0
Use at least 1 L of citrate solution in large glass box to avoid drying.





3. Block in PBST (PBS with 0.3% Triton) with 2% dry milk for 30 min 
Stock: 10X PBS
4. Incubate with primary antibody    at Room temperature .

5. Wash 3 x in PBST.
6. Incubate with appropriate biotinylated secondary (for immunohistochemistry) or fluorescent secondary (for immunofluorescence) antibody at 1:200 in PBST + milk for 60 min  at Room temperature . 
For immunohistochemistry incubate in ABC reagent (Vector) for 60 min  at Room temperature .

7. Rinse 3 x PBST.
8. Mount slides with antifading medium for immunofluorescence or dehydrate through ethanol series and xylenes and mount in Permount for immunohistochemistry.
TITLE: Purification of Lambda Protein Phosphatase
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol details the purification of Lambda protein phosphatase.

[STEPS]
SECTION: Purification procedure
1. To purify Lambda protein phosphatase (λ PPase), fuse the protein phosphatase to a N-terminal 6xHis-tag through cloning into a pET-DUET1 vector (available from Addgene). 

SECTION: Purification procedure
2. After the transformation of the pET-DUET1 vector encoding 6xHis-TEV-λ PPase in E. coli Rosetta pLysS cells (Novagen Cat# 70956-4), grow cells in 2x Tryptone Yeast extract (TY)medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C. 

SECTION: Purification procedure
3. Once the cells reached an OD600 of 0.8, induce protein expression with 100 micromolar (µM) isopropyl β-D-1-thiogalactopyranoside (IPTG) for 960 min  at  18 °C. 

SECTION: Purification procedure
4. Collect the cells by centrifugation and resuspend in lysis buffer, complete EDTA-free protease inhibitors (Roche), CIP protease inhibitor (Sigma), and DNase (Sigma)). 

Lysis buffer:
 
 
SECTION: Purification procedure
6. Clear lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific). 

SECTION: Purification procedure
7. Filter the supernatant through an 0.45 µm filter and load onto a pre-equilibrated 5 ml His-Trap HP column (Cytiva). 

SECTION: Purification procedure
8. After bind His-tagged proteins to the column, wash the column with three column volumes of wash buffer. 

Wash buffer:
 
 
SECTION: Purification procedure
9. Elute the proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM). 

SECTION: Purification procedure
10. Pool and incubate the fractions containing the 6xHis-TEV-λ PPase 60 minwith TEV protease at 4 °C. 

SECTION: Purification procedure
11. After the 6xHis tag was cleaved off, recapture 6xHis tag and His-tagged TEV protease with nickel beads for 60 min at  4 °C. 

SECTION: Purification procedure
12. Pellet the beads by centrifugation and the supernatant, concentrate containing the λ PPase protein using a 30 kDa cut-off Amicon filter (Merck Millipore) and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva). 

SECTION: Purification procedure
13. Elute the proteins with SEC buffer. 

SEC buffer:
 
 
SECTION: Purification procedure
14. Analyse the fractions by SDS-PAGE and Coomassie staining. 

SECTION: Purification procedure
15. Pool the fractions containing purified λ PPase. 

SECTION: Purification procedure
16. After concentrating the purified protein, aliquot the protein and snap-frozen in liquid nitrogen. 

SECTION: Purification procedure
17. Store the proteins at  -80 °C.

SECTION: Purification procedure
5. Sonicate cell lysates twice for 30 s.
TITLE: Utilization Patterns and Trends in The Use of Medications for Asthma Control in a Cohort of Colombian Patients, 2017-2019
AUTHORS: Jorge        Machado Alba

[DESCRIPTION]
Background:Asthma affects approximately 358 million people worldwide.
Objective:To determine the trend in the use of medications used to treat asthma in a group of patients affiliated to the Colombian health system.
Methods:A retrospective study on prescription patterns of medications used to treat asthma in patients over 5 years of age between 2017 and 2019. Sociodemographic variables, medications used and combinations, persistence of use and prescribing physician were considered. Data were obtained from a drug-dispensing database from Colombia. 
Results:10,706 people with a diagnosis of asthma were identified, with predominance in female sex (56.8 %), mean age 32.2 ± 26.1 years. At the start of follow-up, 53.2% of patients aged 5-11 years were receiving monotherapy, mean 1.5 ± 0.6 drugs/patient, especially inhaled corticosteroids (ICS; 55.9%) and short-acting β-agonists (SABA; 55.6%). Moreover, in patients older than 12 years, 53.5% were treated in monotherapy, mean 1.6 ± 0.7 drugs/patient, of which 45.9% were on SABA, and 37.1 % on ICS.Between 63.0% and 83.6% of patients were treated by a general practitioner. The proportion of patients on the ICS/ long-acting β-agonists (LABA) combination grew at 24 months follow-up by 411% and 177%, respectively. 12.5% of patients (n=495) received triple therapy (ICS/LABA+LAMA [long-acting anticholinergics]), particularly with fluticasone/salmeterol + tiotropium. 
Conclusions:Patients with a diagnosis of asthma older than 5 years in Colombia are mainly receiving control therapy with ICS, alone or combined with SAMA and, to a lesser extent, with LABA.

[STEPS]