eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Aims: Stress granules (SGs) are transient cytoplasmic mRNA and protein complexes that form in eukaryotic cells under stress. SGs are related to multiple diseases, but there are no reports of the existence of SGs in atrial fibrillation (AF); hence, the effects of SGs in AF are still unknown. <br />Methods and results: Cell models of AF were established by field stimulation at 600 times per minute. HL-1 cells, cardiomyocytes and cardiac fibroblasts were transfected with pcDNA and G3bP1-cDNA plasmid by Lipofectamine 2000. The presence of SGs was detected by immunofluorescence staining against GTPase-activating protein SH3 domain binding protein 1 (G3BP1) and poly(A)-binding protein 1 (PABP-1) and electron microscopy. Stable HL-1 cell lines transfected with lentivirus overexpressing G3BP1 (pEGFP-LV-G3BP1) were constructed to further investigate the role of SGs in AF. The effects of SGs on reactive oxygen species (ROS) and calcium overload in tachypaced HL-1 cells were studied by flow cytometry. The effects of SGs on cardiac fibroblast proliferation and the protein expression level of collagen I/III and fibronectin-1 were also studied. Here, we first showed that SGs detected by G3BP1 and PABP-1 are present and markedly elevated in both tachypaced mouse cardiomyocytes and HL-1 atrial cells. The presence of SGs in tachypaced atrial myocytes was also confirmed by electron microscopy. SG formation in HL-1 cells with G3BP1 overexpression significantly inhibited the rapid pacing-induced increase in ROS level and attenuated calcium overload (all P&lt;0.05). Furthermore, SGs inhibited cardiac fibroblast proliferation (P&lt;0.05) and decreased the protein expression level of collagen I/III and fibronectin-1 in cardiac fibroblasts stimulated by angiotensin II (all P&lt;0.05). <br />Conclusions: SGs are rapidly induced and present in AF, and upregulation of SGs confers cytoprotection against oxidative stress, calcium overload and atrial fibrosis in AF. </p>

[STEPS]
TITLE:         ELISA for quantification of tumor necrosis factor alpha (TNF-α) in human serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Tumor necrosis factor</span><span> (</span><span style = "font-weight:bold;">TNF)</span><span style = "font-weight:bold;font-style:italic;"> </span><span>is a cell signaling cytokine involved in systemic inflammation. It is one of the proteins that make up the acute phase reaction [1].</span></div><div class = "text-block">Reference</div><div class = "text-block"><span>1. Pasquereau S, Kumar A, Herbein G. Targeting TNF and TNF Receptor Pathway in HIV-1 Infection: from Immune Activation to Viral Reservoirs.</span><span style = "font-style:italic;">Viruses</span><span>. 2017;9(4):64. Published 2017 Mar 30. doi:10.3390/v9040064</span></div></div>

[STEPS]
?. An anti-human TNF-α 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 TNF-α 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-TNF-α 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-TNF-α 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 TNF-α 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 TNF-α standard dilutions and the human TNF-α sample concentration is determined.
?. For better results place the microplate on a microplate shaker in every incubation.
TITLE: Artificial Cerebrospinal Fluid IV (ACSF.IV)
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes making Artificial Cerebrospinal Fluid IV (ACSF.IV). ACSF.IV is used for multiple applications including incubation of fresh mouse brain slices prior to electrophysiological recording.</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: No Title
AUTHORS: 

[DESCRIPTION]
<p>Adapted from Qiagen's <a href="https://www.qiagen.com/us/resources/resourcedetail?id=d2b85b26-16dd-4259-a3a7-a08cbd2a08a3&amp;lang=en" target="_blank" rel="noopener noreferrer">genomic DNA handbook</a> and the <a href="https://www.qiagen.com/us/resources/resourcedetail?id=b45c3cc3-7f2b-4f4a-aa37-21d814ed3730&amp;lang=en" target="_blank" rel="noopener noreferrer">user protocol for mosquito DNA extraction.</a></p>

[STEPS]
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TITLE: Low-input DNA extractions in 96-well plates
AUTHORS: Tom Harrop, Reuben McKay Vercoe, Ciarán Cuddy

[STEPS]
?. [Prepare lysate for extraction]
Prepare lysate for extraction
?. [Prepare lysate for extraction]
Dissect e.g. 1 M. hyperodae pupa for a single sample.Place up to 88 individuals in a 1000 µL, round well deepwell plateAdd 2 3.2 mm stainless steel ball bearings and 100 µL of lysis buffer with RNase to each well.
?. [Prepare lysate for extraction]
Homogenise the tissue for 90 seconds at 1200 RPM using a plate-compatible tissue homogenizer, e.g. SPEX SamplePrep 2010 Geno/Grinder.
?. [Prepare lysate for extraction]
Prepare 100 µL lysis buffer per sample.  AB1Lysis solution99 µL2RNase solution (10 mg/mL)1 µL3Total100 µLThe Zymo lysis solution can be bought separately.This protocol also works with the Zymo Solid Tissue Buffer II that is supplied with the Quick-DNA Magbead Plus kit. Solid Tissue Buffer II comes as a 2x concentrate and has to be diluted with nuclease-free water.
AB1Lysis solution99 µL2RNase solution (10 mg/mL)1 µL3Total100 µL
?. [Prepare lysate for extraction]
Seal the plate and mix on the plate shaker. Make sure the paste is resuspended.Incubate at 37°C for 30 minutes.
37 °C
?. [Prepare lysate for extraction]
Add 5 µL Proteinase K solution (20 mg/mL) and mix on the plate shaker.Incubate at 55°C for 120 minutes, shaking the plate for one minute every 30 minutes.
55 °C
?. [Prepare lysate for extraction]
Spin the plate for 10 minutes at 1,000 RPM.
Centrifuge: 1000 33, 10 min, 15 10
?. [Prepare lysate for extraction]
Transfer 100 µL of lysate to a 0.8 mL deepwell plate.
?. [DNA extraction]
Set up the reagents from the ZYMO Quick-DNA Magbead Plus Kit
?. [DNA extraction]
Add the following reagents to the 7-position ReservoirRack  ABCDEFGHIJKLM1ReagentReservoir
volume (mL)81624324048566472808821: 300 µL MBB : 5 µl beads3026455085752599651240514845172851972522165246052704532: DNA pre-wash buffer10033405340734093401134013340153401734019340213402334043: g-DNA wash buffer1005340934013340173402134025340293403334037340413404534054: Tris-HCl10702902110213021502170219022102230225022702
ABCDEFGHIJKLM1ReagentReservoir
volume (mL)81624324048566472808821: 300 µL MBB : 5 µl beads3026455085752599651240514845172851972522165246052704532: DNA pre-wash buffer10033405340734093401134013340153401734019340213402334043: g-DNA wash buffer1005340934013340173402134025340293403334037340413404534054: Tris-HCl10702902110213021502170219022102230225022702
?. [DNA extraction]
Run the epMotion protocol 88x Quick-DNA Magbead Plus Low Volume 40_6.
?. [DNA extraction]
Run the protocol with level sensing enabled.
TITLE: Intramuscular Injection Adult Mouse
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
This protocol describes the general procedures used for intramuscular injection in adult mice.  

Note: 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.

[STEPS]
TITLE: Japanese encephalitis virus isolation
AUTHORS: Wenjing  Liu, Shihong Fu

[DESCRIPTION]
The mosquito grinding supernatant and CSF samples were inoculated into Vero cells to finish virus isolation.

[BEFORE_START]
Mosquito grinding supernatant was sterilized using a filter with a pore size of 0.22 μm.

[STEPS]
SECTION: virus isolation
1. The filtered sterile mosquito grinding solution was inoculated into 24-well plates grown to 80% monolayer Vero cells, 70 μl of each well was added for virus isolation, and a 3-well blank control was set for each plate.(for CSF samples 100μl of each well was added for virus isolation).
SECTION: virus isolation
2.  Incubate at 37℃ for 1 h.
SECTION: virus isolation
3.  1 ml of MEM medium containing 2% FBS was added to each well for maintenance of growth. Cultivate at 37℃ 5% CO2 and observe under microscope every day whether cytopathic effect (CPE) occurs.
SECTION: virus isolation
4. Specimens with no CPE were used for blind transmission for three generations .
TITLE: Dissection and immunohistochemistry of mouse vagal ganglia
AUTHORS: Thomas Taylor-Clark, Seol-Hee Kim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Mice are euthanized, perfused with fixative and the vagal ganglia extracted. Vagal ganglia are then cyosectioned. Slices are stained for protein expression using immunohistochemistry. Expression of specific proteins and reporter proteins isarevisualized using microscopy.</div></div>

[STEPS]
?. 6 to 8 weeks old mice (male) were euthanized by CO2 inhalation.
?. Mice were transcardially perfused with phosphate buffered saline (PBS) to remove blood followed by 3.7% formaldehyde (PFA) in PBS.
?. Vagal ganglia (including both jugular and nodose ganglia) were collected and fixed with 3.7% PFA for 2 hours on ice.
?. Vagal ganglia were transferred to 18% sucrose solution for overnight at 4 °C.
?. Tissue were mounted in optimal cutting temperature compound and frozen
?. Tissue were cut at 20 µm thickness and collected onto superfrost plus microscopy slide glass
?. Tissue were washed with PBS three times for 10 min.
?. Tissue were blocked with blocking buffer (1% bovine serum albumin/10% normal donkey serum/0.3% triton x-100 in PBS) for 45 min at room temperature.
?. Tissue were incubated primary antibodies diluted in blocking buffer overnight at 4 °C.      Set 1. Rabbit anti-TrkA (1:300, 06-574, Millipore) and Goat anti-VR1 (1:150, SC-12498, Santa Cruz)      Set 2. Rabbit anti-TrkA (1:300, 06-574, Millipore) and Goat anti-TrkB (1:300, AF1494, R&D Systems)
?. Tissue were washed with 0.2% tween20 in PBS (PBST) for 10 min for three times at room temperature.
?. Secondary antibodies were diluted in the 1% bovine serum albumin/5% normal donkey serum/0.2% PBST and tissue were incubated with it for 1 hour at room temperature.      1. Alexa Fluor 488 donkey anti-rabbit (1:300, A21206, Invitrogen)      2. Alexa Fluor 647 Chicken anti-Goat (1:300, A21469, Invitrogen)
?. Tissue were washed with 0.2% tween20 in PBS (PBST) for 10 min for three times at room temperature.
?. Tissue were cover-slipped with DPX for imaging and left to dry in the dark overnight.
TITLE: NMJ
AUTHORS: Jake Willows, Kristy Townsend, Magdalena Blaszkiewicz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">A series of protocols to fluorescently label neuromuscular junctions in mice for qualitative and quantitative studies.</div></div>

[STEPS]
?. See attachments under Abstract for PDF copies of protocols.
TITLE: High Resolution Ex Vivo Structural MRI Imaging &amp; Analysis Protocol
AUTHORS: Bruce  Fischl, Andre  Van Der Kouwe, Divya  Varadarajan, Matthew  Dylan Tisdall, Leah  Morgan, Robert  Frost, Allison  Stevens

[DESCRIPTION]
At the Martinos Center for Biomedical Imaging, we are continuously developing cutting-edge techniques to enable high-resolution ex vivo MR Imaging data and novel data processing techniques. We form one site of a multi-site project with the goal to perform an imaging & analysis pipeline towards generating a cell census for Brodmann's Area 44/45 in the human brain. Magnetic resonance imaging (MRI) is used to establish a macroscopic reference coordinate system of laminar and cytoarchitectural boundaries. Cell counting is obtained with both traditional immunohistochemistry, to provide a stereological gold-standard, and with a custom-made inverted confocal light-sheet fluorescence microscope (LSFM) for 3D imaging at cellular resolution. Finally, mesoscale optical coherence tomography (OCT) enables the registration of the distorted histological cell typing obtained with LSFM to the MRI-based atlas coordinate system. This protocol outlines the steps involved in generating the MRI data for a reference system of the cell census.

[STEPS]
TITLE:   Enzyme linked immunosorbent assay for investigating the binding of chemically prepared protein-LAG-anti-IgY (SpLAG-anti-IgY) to avian and mammalian immunoglobulins. 
AUTHORS: Angel  Justiz-Vaillant, Norma McFarlane-Anderson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This SpLAG-anti-IgY ELISA can be used to detect specific antibodies in various animal species including human, goat, donkey, mouse, rat, dog, cow, horse, ostrich, duck, pigeon, bantam hen, rabbit, chicken, monkey, pig, hamster, and many birds, wild and zoo animals species [1].</div><div class = "text-block">1. Vaillant AJ, McFarlane-Andersonv N, Wisdom B, Mohammed W, Vuma S, et al. (2013) Immunoglobulin-binding Bacterial Proteins (IBP) Conjugates and their Reactivity with Immunoglobulin in Enzyme-Linked Immunosorbent Assays (ELISA). J Anal Bioanal Tech 4: 175. doi:10.4172/2155-9872.1000175</div></div>

[STEPS]
?. This ELISA is used to study the interaction of protein-LAG-anti-IgY (SpLAG-anti-IgY) with different immunoglobulin preparations from avian and mammalian species.
?. The 96 well microtitre plate is coated overnight at 4°C with 2 µg/µl per well of a mixture of SpL, SpA, SpG and anti-IgY (equal concentration of each protein) in carbonate-bicarbonate buffer pH 9.6.
?. Then plate is treated with bovine serum albumin solution and washed 4X with PBS-Tween.
?. 50 µl of  animal serum (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 SpLAG-anti-IgY conjugate diluted 1:5000 in PBS-non-fat milk is added to each well and  incubated for 1h at RT. The plate is washed 4X with PBS-Tween.
?. 50 µl of 4 mg/ml o-phenylenediamine solution (OPD) is added and the plate is incubated 15 minutes at RT in the dark.
?. The reaction is stopped with 50 µl of 3M H2SO4 solution.
?. The plate is visually assessed for the development of colour and read in a microplate reader at 492 nm.
?. A cut-off point should be calculated as the mean of the optical density of negative controls x 3. The higher the OD value the higher will be the affinity of SpLAG-anti-IgY to avian and mammalian immunoglobulins.
TITLE: Synthetic Leaching Procedure for Soils
AUTHORS: joshua.padilla

[DESCRIPTION]
This is a protocol following the EPA's Synthetic Leaching Procedure (SLP) for soils. This method provides an estimate of rainfall-leachable ions from soils, particularly from those that are metal-contaminated. 

[STEPS]
SECTION: Preparation of SLP acid mixture.
1. Add 2.4 g of H2SO4 and 1.6 g of HNO3 to about 30 mL of DI water in a graduated 50 mL centrifuge tube. Adjust the final volume to 40 mL. Add 1 mL of this to about 40 mL of DI water in a second graduated 50 mL centrifuge tube. Adjust the final volume to 50 mL.  Wear proper PPE at all times.
SECTION: Preparation of SLP extractant.
2. Calibrate a pH meter. 
SECTION: Preparation of SLP extractant.
3. Fill a beaker with a volume of DI water sufficient for your needs. Add a stir bar to the beaker and place it on a stir plate. Insert the pH meter into the the DI water and turn the stir plate on. 
SECTION: Preparation of SLP extractant.
4. Using a disposable transfer pipette, add your SLP acid mixture (step 1) dropwise to the stirring DI water. Add one or two drops at a time and wait for the pH reading to stabilize. If you are extracting soils from east of the Mississippi River, adjust the final pH to 4.2. For soils west of the Mississippi River, adjust to the final pH to 5.0. This should require 10-15 drops of the SLP acid mixture for about 1 L of extractant. 
SECTION: SLP Extraction
5. SLP extractions are done at a soil:solution ratio of 1:20. I typically use 2g:40 mL, however, for biochar screening experiments, I'll use 40g:800 mL in 1L bottles. Weigh soil into a properly sized vessel with a secure lid. Add SLP extract at a volume that is 20x the mass of soil. Cap the vessel and transfer to a platform shaker for 18 hours. (I usually start these at 4:00 PM and take them off at 10:00 AM the next morning.) 
SECTION: SLP Extraction
6. Filter the liquid through a 0.45 um filter. This can be analyzed for solutes of interest according to the proper method.
TITLE: Bacterial fluorescent in situ hybridization (FISH) in Ephydatia muelleri
AUTHORS: Haley Womack, Gwendolyn Geiger, Scott Nichols

[DESCRIPTION]
This protocol is used to visualize bacteria in and around gemmule-hatched freshwater sponges using a Eubacteria FISH probe. 

[STEPS]
SECTION: Grow sponges
1. Add 3-4 mL mL of spring water to a 35mm coverslip bottom dish and place 1-3 gemmules into the center of the inner well.

SECTION: Grow sponges
2. Let grow in the dark (to reduce autofluorescence of algae) at Room temperature for about 10080 min (1 week) or until tissue appears developed.

SECTION: Day One
3. Make fixative of 4% formaldehyde in 95% EtOH
150 µL  
 

SECTION: Day One
4. Remove water from the outer well area, add 150 µL of fixative to the sponge, and let sit at room temperature for 240 min 
 


SECTION: Day One
5. Closer to the end of the incubation, prepare a 0.1% solution of Tween-20 in 1X PBS (now referred to as PBS-Tw) 
12 mL 
Prepare 10% formamide in 2X SSC
2 mL 

 


SECTION: Day One
7. Remove the PBS-Tw and repeat the wash two more times
SECTION: Day One
6. Remove the fixative from the outer well area and replace with 4 mL  of PBS-Tw to each well
SECTION: Day One
8. Add 2 mL of freshly prepared 10% formamide in 2X SSC to each sample and incubate at 37 °C for 120 min 
SECTION: Day One
9. Prepare 1:100 eub338 probe [1µM final concentration] in Hybridization Buffer (Molecular Instruments) and heat to 37℃
 

SECTION: Day One
10. Remove the 10% formamide in 2X SSC from the samples and add 80-100 µL of probe mixture to the inner well, being careful to leave the samples flat
SECTION: Day One
11. Place lid on sample and leave in humid chamber    at 37℃
 

SECTION: Day Two
12. Prepare 10% formamide in 2X SSC
3 mL 
Prepare 2X SSC
3 mL 
Prepare 0.2X SSC 
12 mL 
Warm all solutions to 37 °C in a water bath
 

SECTION: Day Two
13. Remove probe mixture from samples and add 3 mL of pre-warmed 10% formamide in 2X SSC. Incubate for 10 min at 37 °C 
SECTION: Day Two
14. Remove the 10% formamide in 2X SSC from the wells and replace with 3 mL of pre-warmed 2X SSC. Incubate for 10 min at 37 °C 
SECTION: Day Two
15. Remove the 2X SSC from the wells and replace with 3 mL of pre-warmed 0.2X SSC. Incubate for 15 min at 37 °C 
SECTION: Day Two
16. Repeat step 15 two more times, for a total of three washes
SECTION: Day Two
17. Prepare a 1:200 dilution of Hoechst (stock concentration of 10mg/mL) in 0.2X SSC
100 µL 
SECTION: Day Two
18. Remove 0.2X SSC from wells and add about 100 µL of Hoechst solution to each inner well. Incubate for 15 min at Room temperature 
SECTION: Day Two
19. Wash samples with 3mL of 0.2X SSC
SECTION: Day Two
20. Remove wash and add mounting medium

SECTION: Day Two
21. Samples are ready to be imaged, or can be left in the dark at 4 °C for preferably up to a week
TITLE: Coating superfrost microscope slides with gelatin-chromium potassium sulfate
AUTHORS: Benjamin Trist, Alejandra Rangel, Rain Kwan

[DESCRIPTION]
This protocol describes how to coat microscope slides with gelatin-chromium potassium sulfate (gelatin-chrom alum) in preparation for histology or immunohistochemical analysis of thin tissue sections. Slides coated with gelatin-chrom alum exhibit much better retention of tissues mounted after free-floating immunohistochemical staining, especially during alcohol and xylene dehydration steps immediately prior to mounting media embedding and cover slipping.

[STEPS]
SECTION: Experimental Outline
1. Place heated magnetic stirrer in fume hood.
SECTION: Experimental Outline
2. Pre-heat oven to 42 °C.
SECTION: Experimental Outline
3. Heat 1 L dH20 to 50 °C - 60 °C and completely dissolve 10 g gelatin with aid of magnetic stirrer.
SECTION: Experimental Outline
4. Add 1 g chromium potassium sulfate - solution should turn a pale green/blue and be completely clear.
SECTION: Experimental Outline
5. Once dissolved reduce temperature to 40 °C-50 °C.
SECTION: Experimental Outline
6. Place slides for coating into designated slide coating rack(s).
SECTION: Experimental Outline
7. Dip rack of slides into warm (40 °C-50 °C) gel mixture for approximately 20 s-30 s.

SECTION: Experimental Outline
8. Shake the excess liquid from the rack.
SECTION: Experimental Outline
9. Repeat steps 7 and 8.
SECTION: Experimental Outline
10. Place slide rack(s) to dry in the oven at 42 °C for 2880 min.
SECTION: Experimental Outline
11. Store slides in dust-free slide storage box.
SECTION: Experimental Outline
12. Discard Gelatin-Chrom Alum solution into appropriate waste container.
TITLE: Starting a MinION sequencing run using MinKNOW
AUTHORS: Muhammad Faisal, Olin Silander, Nikki Freed

[STEPS]
?. If required plug the MinION into the computer and wait for the MinION and flowcell to ben detected.
?. Choose flow cell 'FLO-MIN106' from the drop-down menu.
?. Then select the flowcell so a tick appears.
?. Click the 'New Experiment' button in the bottom left of the screen.
?. On the New experiment popup screen, select the running parameters for your experiment from the individual tabs:Experiment: Name the run in the experiment field, leave the sample field blank.Kit: Selection: Select RBK004 Run Options: Set the run length to 6 hours (you can stop the run once sufficient data has been collected as determined using RAMPART).Basecalling:  Select 'fast basecalling'.Output: The number of files that MinKNOW will write to a single folder. By default this is set to 4000 but can be reduced to make RAMPART update more frequently.Click 'Start run'.
?. Monitor the progress of the run using the MinKNOW interface.