eamag/protocols-with-steps huggingface dataset:
TITLE: Purification of the any avian IgY using chloroform, trichloroacetic acid and affinity purification using HiTrap™ Columns.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This IgY purification technology can be used to purified IgY from the egg yolk of any bird as ducks, bantam hens, ostrich, chicken, pigeon, pheasant, quail, guinea hen, cattle egret and goose.</div></div>

[STEPS]
?. The IgY fraction of bantam hen was isolated by the chloroform-Trichloroacetic acid (TCA) -affinity chromatography method.
?. The egg is washed with warm water and the egg yolk is separated from the egg white.
?. The membrane is broken and the egg yolk collected.
?. The egg yolk is then diluted 1:3 in phosphate buffered saline (PBS), pH 7.4.
?. To 1/3 of the egg yolk mixture an equal volume of chloroform is added,
?. The mixture is then shaken and centrifuged for 30 min (1000×g, RT).
?. The supernatant is decanted and drop 1 ml of cold TCA and let the sample stand for 30 min at RT.
?. The mixture is then centrifuged as previously described.
?. The precipitate containing IgY is dissolved in PBS (pH 7.4) at a volume equivalent to 1/6 of the original volume of the egg yolk.
?. After that the preparation is dialyzed against 1L of PBS (pH: 7.4 for 24 h at 4°C).
?. The IgY is then removed from the dialysis tubing.
?. IgY concentration is assessed by an available methodology.
?. IgY samples then are further purified by affinity chromatography.
?. The preparation can be stored at –20°C.
?. By a sandwich ELISA the IgY concentration can be assessed.
?. Use Affinity Purification Using HiTrap™ Columns as follows:
?. Fill the syringe with distilled water.
?. Connect the column to the syringe
?. Snap off the tab on the column outlet.
?. Wash out the ethanol with distilled water.
?. Equilibrate the column with binding buffer.
?. The recommended ﬂow rate is 5 ml/min*.
?. Apply the sample using a syringe linked or ﬁtted to Luer connector
?. Use a ﬂow rate of 1 to 5 ml/min during sample application
?. Wash with at least 51 ml of binding buffer.
?. Maintain a ﬂow rate of 10 ml/min for washing.
?. Elute with 50 ml of elution buffer.
?. After elution, regenerate the column by using wash buffer
?. Re-equilibrate the column with 26 ml of binding buffer.
?. Store it until a new purification
TITLE: Neuromelanin-positive Neuron Density in Substantia Nigra Image Analysis
AUTHORS: Hemanth Ramesh Nelvagal, Toby J Curless, Zane Jaunmuktane

[DESCRIPTION]
The protocol covers the steps to measure neuromelanin-positive neuron density in substantia nigra using image analysis tools including NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer, Cellpose and QuPath.

[STEPS]
SECTION: Annotation and Deconvolution
1. Manually annotate the substantia nigra on NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer.

SECTION: Annotation and Deconvolution
2. Download the annotations using a Python script, and then import into QuPath [1] using a Groovy script
SECTION: Segmentation and Calculating Neuromelanin-positive Cell Density
3. Segment neuromelanin cells using Cellpose [2,3] via the QuPath Cellpose extension [4], followed by an object classifier to filter out non-specific detections. 
SECTION: Segmentation and Calculating Neuromelanin-positive Cell Density
4. Calculate neuromelanin-positive cell density by the number of neuromelanin-positive cells divided by the area of the region of interest (neuromelanin-positive cells per mm^2).
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: CD34+ isolation from human bone marrow
AUTHORS: Mohsen Khosravi-Maharlooei, Markus Holzl, Austin Chen, Megan Sykes

[DESCRIPTION]
This protocol details the steps for isolating CD34+ cells from human bone marrow. The CD34+ cells isolated from this protocol can be used for generating humanized mice through reconstitution of immune cells via IV injection after bone marrow ablation. These cells can also be used for mixed lymphocyte reaction experiments.



 


[BEFORE_START]
Human bone marrow is a rich source for CD34+ hematopoietic stem cells. CD34+ cells can be easily isolated and further processed.


[STEPS]
1. Transfer the content of the collection bag into a sterile flask
2. Add 250 mLBM Medium to the bag and rinse it thoroughly

3. Transfer the content of the collection bag into the sterile flask
4. Layer 35 mLof the suspension over 15 mL of Histopaque

5. Centrifuge the tubes for 30 minutes 500 g without brake at RT

6. Collect the leukocyte ring in 50 mL Falcons and fill up with BM medium

7. Wash once by centrifuging 6 minutes 500 g 

8. Resuspend the cells in MACS buffer and count (take 50 µLfor FACS confirmation = PRE)

9. Wash down again and resuspend the pellet according to the protocol (130-046-702 MACS Human CD34+ kit)
10. Add 300 µL of MACS buffer per 10^8 cells

11. Add 100 µL of FcR-B reagent per 10^8 cells, mix it and incubate in fridge for 15 minutes

12. Add 100 µLof CD34 beads per 10^8 cells to the suspension and mix and let it sit for 30 min (fridge)

13. Fill up with 50 mL MACS buffer and strain through a blue strainer (40 uM)

14. Wash cells (500 g6 min) and resuspend in MACS buffer 500 uL/200,000,000 cells. If you have more cells, increase volume accordingly. E.g 3 *10^9 cells = 7,5mL. Aliquot this volume to more than one (with 3 mL prerinsed) MACS column.

15. Wash with buffer 3 mL 3 times and keep negative fraction (Take 50 µL for FCM = POST neg)

16. Put the column out of the magnet and push out positive fraction with 5 mL Buffer and the plunger

17. Collect the positive fractions. (Take 50 µL for FCM = Post pos)

18. Process the cells as desired (injection in mouse or cryopreservation)
19. Check the puritiy with FACS
20.
TITLE: Single worm lysis
AUTHORS: Ida Barlow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Exraction of genomic DNA (gDNA) from single adult </span><span style = "font-style:italic;">C. elegans </span><span>suitable for downstream molecular biology applications such as PCR.</span></div></div>

[STEPS]
?. Prepare 1mL 1X PCR buffer with 0.4mg/ml Proteinase K:nb. volumes can be scaled as required
[water]
[10X PCR buffer (pH8.3)]
[20mg/ml Proteinase K solution]
[Total volume]
10X PCR buffer (pH8.3) preparation:AutoclaveCool (temperature affects pH)pH meter and add hydrochloric acid to achieve the correct pH
?. Prepare worm lysis tubes with PCR buffer and allow   per worm.Pipette buffer into lid of PCR tube (up to 45ul can fit into tube lid)
[1X PCR buffer + PK]
?. Pick a single worm at a time into the liquid in the 200uL PCR tube. Make sure you can see the worm swimming in the buffer
?. Close the lids onto the tubes of the 200uL tube and spin in a microfuge so that the liquid and worms are at the bottom of the tube
?. Burst the cells open by freezing the tubes in the -80oC freezer for 10 minutes
?. Transfer tubes to thermocyler and incubate on worm lysis program:60oC - 1hr 10 mins95oC - 15 mins
?. Store gDNA in -20oC freezer until required. Use 2ul per 25uL PCR reaction.
TITLE: Golden Gate plasmids used for transfection of Perkinsus marinus
AUTHORS: Elin Einarsson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">We have created plasmids for transfection of the parasite Perkinsus marinus using Golden Gate cloning. We have explored the possibility to either FACS sort (GFP/mCherry fused to MOE membrane protein) or using drug selection (bleomycin, blasticidin S or puromycin) to enrich transfected cells. </div></div>

[STEPS]
?.
TITLE: Polson method for IgY isolation with modification. 
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Immunoglobulin Y (IgY) is the mayormolecule present in the avian egg yolk. This protein functions as antibody and fulfils important functions in the protection of the embryo against challenging stimuli [1]. IgY has been isolated from the egg yolk of many birds [1]. These antibodies are important therapeutically, but also they have  been used as immunological tool in many immunodiagnostic assays.</div><div class = "text-block">Much time IgY concentration is calculated from the water soluble fraction (WSF), which means that there is not need of treating the preparation with alcohol to separate the protein pellet. </div><div class = "text-block">Reference</div><div class = "text-block">1. Justiz Vaillant AA, Ramirez N, Cadiz A, Ferrer B, Akpaka P, et al. (2013) Separation and Reactivity of Avian Immunoglobulin Y. J Chromat Separation Techniq 4: 173. doi:10.4172/2157-7064.1000173</div></div>

[STEPS]
?. Wash the egg with warm water and crack the eggshell carefully.
?. Separate the egg yolk from the egg white manually and place the egg content on a tissue paper to help to remove as much egg white as possible.
?. Wash four times the egg yolk with 100 ml of PBS, pH 7.4.
?. Break the egg yolk membrane and pour it into a 50 ml tube. Then, dilute egg yolk 1:5 in PBS, pH 7.4, and add an equal volume of chloroform (ACS reagent grade, Sigma-Aldrich).
?. Shake the mixture, and vortex and roll the tube containing the mixture on rolling mixer for 15 minutes.
?. Centrifuge the mixture for 15 min (2000×g at 18°C).
?. Decant  the supernatant, which contains the water-soluble fraction (WSF) that is the diluted egg yolk plasma, rich in IgY (25 mg/ml), that is
?. approximately four times the IgY concentration present in the serum (6 mg/ml).
?. Decant the supernatant, which contains the water-soluble fraction (WSF) that is the diluted egg yolk plasma, rich in IgY (25 mg/ml), which is approximately four times the IgY concentration present in the serum (6 mg/ml).
?. 
?. Precipitate egg yolk antibodies (IgY) by dropping the WSF with cold ethanol,  trichloroacetic acid (TCA) or the classic polyethylene glycol. (PEG).
?. Estimate the protein concentration.
?. 
?. Run a protein gel electrophoresis (PAGE) of the IgY to assess the molecular weight of the chicken egg yolk antibody, protocol for this can be found elsewhere.
?. Then use Phosphate-buffered saline (PBS) pH=7.4 to dialyze the preparations for 8 hrs at 4°C.
?. The dialysate is stored at -20°C until further use.
TITLE: Adapter ligation with AMII
AUTHORS: Josh Quick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a subprotocol for performing adapter ligation with AMII</div></div>

[STEPS]
?. Set up the following AMII adapter ligation reaction:Component                                                                    VolumeEnd-repaired amplicon pools                                      NEBNext Quick Ligation Reaction Buffer (5X)         Adapter Mix (AMII)                                                       Quick T4 DNA Ligase                                                   Total
30 µl
10 µl
5 µl
5 µl
50 µl
There will be some variation in clean-up efficiencies but expect to carry around 80% through a clean-up.
?. Incubate at room temperature for
?. Add (1:1) of SPRI beads to the sample tube and mix gently by either flicking or pipetting.
50 µl
Vortex SPRI beads thoroughly before use to ensure they are well resuspended, the solution should be a homogenous brown colour.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Incubate for  at room temperature.
?. Place on magnetic rack and incubate for  or until the beads have pelleted and the supernatant is completely clear.
?. Carefully remove and discard the supernatant, being careful not to touch the bead pellet.
?. Add  SFB and resuspend beads completely by pipette mixing.
250 µl
SFB will remove excess adapter without damaging the adapter-protein complexes. Do not use 70% ethanol as in early clean-ups.
?. Pulse centrifuge to collect all liquid at the bottom of the tube.
?. Remove supernatant and discard.
?. Repeat steps 14-16 to perform a second SFB wash.
?. Pulse centrifuge and remove any residual SFB.
You do not need to allow to air dry with SFB washes.
?. Add EB and resuspend beads by pipette mixing.
15 µl
?. Incubate at room temperature for .
?. Place on magnetic rack.
?. Transfer final library to a new 1.5mL Eppendorf tube.
TITLE: RNAscope pretreatment optimized for fixed frozen retinas
AUTHORS: Grace Burgin, Inbal Benhar

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a pretreatment for RNAscope optimized for fixed frozen retina sections </div><div class = "text-block">(emphasizes adhesion of tissue to the slides) </div></div>

[STEPS]
TITLE: Immunohistochemical staining, vibratome sections
AUTHORS: Joris Van Asselberghs, Veerle Baekelandt

[DESCRIPTION]
This protocol outlines general procedures for Immunohistochemical staining, using vibratome sections. 

[BEFORE_START]
This protocol uses floating sections in 24-well plate in 1 X.

[STEPS]
SECTION: Quench Endogenous Peroxidase Activity
1. Incubate samples in 1 x PBS 3% H2O2, for 10 min at Room temperature on the wobbler (to quench endogenous peroxidase activity) using 500 µL / well.

SECTION: Quench Endogenous Peroxidase Activity
2. Rinse with 0.1 % (v/v).

SECTION: Quench Endogenous Peroxidase Activity
3. Rinse with 0.1 % (v/v) for 5 min on wobbler. 

SECTION: Quench Endogenous Peroxidase Activity
4. Repeat: rinse with 0.1 % (v/v) for 5 min on wobbler. 

SECTION: Primary Antibody Incubation
5. Add 250 µL (accordingly to the secondary Abs)  40 min at Room temperature on wobbler.
 

SECTION: Primary Antibody Incubation
6. Rinse with 0.1 % (v/v).
SECTION: Primary Antibody Incubation
7. Rinse with 0.1 % (v/v) for 5 min on wobbler. 
SECTION: Primary Antibody Incubation
8. Repeat: rinse with 0.1 % (v/v) for 5 min on wobbler. 
SECTION: Secondary Antibody Incubation
9.  Add 250 µL for 30 min at Room temperature on wobbler. 
 

SECTION: Secondary Antibody Incubation
10. Rinse with 0.1 % (v/v).
SECTION: Secondary Antibody Incubation
11. Rinse with 0.1 % (v/v) for 5 min on wobbler. 
SECTION: Secondary Antibody Incubation
12. Repeat: rinse with 0.1 % (v/v) for 5 min on wobbler. 
SECTION: Streptavidine-HRP complex Incubation
13. Add 250 µL for 30 min at Room temperature on wobbler. 
 

SECTION: Streptavidine-HRP complex Incubation
14. Rinse with 0.1 % (v/v).
SECTION: Streptavidine-HRP complex Incubation
15. Rinse with 0.1 % (v/v) for 5 min on wobbler. 
SECTION: Streptavidine-HRP complex Incubation
16. Repeat: rinse with 0.1 % (v/v) for 5 min on wobbler. 
SECTION: DAB + H2O2
17. 
 

Prepare DAB solution: 10 mg for 25 mL; dissolve and filter through 0.22 µm filter, add H2O2 just before use! Or Vector SG (TH).
(Add 1.4 µL for 5 mL).
SECTION: DAB + H2O2
18. Add 250 µL (ON DAB BENCH!). Allow reaction to proceed for a few minutes at Room temperature without wobbling. 
SECTION: DAB + H2O2
19. Rinse with 0.1 % (v/v).

SECTION: DAB + H2O2
20. Replace TBS Triton X-100 with PBS or 0.1 % (v/v) in case sections are not to be mounted immediately. Store at 4 °C.
SECTION: ½ PBS + ½ AD Rinse
21. Briefly rinse sections in ½ PBS + ½ AD and mount on gelatin coated microscopy slides. Allow to dry for 30 min in flow or couple of hours on bench.

SECTION: Dehydration
22. Dehydrate samples in 70 % (v/v) for 5 min. 
SECTION: Dehydration
23. Dehydrate samples in 90 % (v/v) for 5 min. 
SECTION: Dehydration
24. Dehydrate samples in 100 % (v/v) for 5 min. 
SECTION: Dehydration
25. Repeat: dehydrate samples in 100 % (v/v) for 5 min.
SECTION: Dehydration
26. Replace ethanol with Histoclear II for 5 min.
SECTION: Mounting
27. Mount coverslips on top of the slides with DPX and allow to dry 30 min in the flow. 
SECTION: Mounting
28. Press out bubbles next morning.
TITLE: Fluorescent Immunolabelling for Alpha-Synuclein in neuronal primary culture  (Testing PFF toxicity)
AUTHORS: Sabina Marciano, Rong Chen, Ted Dawson, Roberta Marongiu

[DESCRIPTION]
This protocol is designed to perform fluorescent immunolabelling on neuronal primary culture after PFF incubation.
The labelling of phosphorylated Alpha-synuclein is considered as a marker of the PFF toxicity and should be performed to test the PFF before in vivo injection.

See protocol for PFF preparation 
https://www.protocols.io/view/production-of-alpha-synuclein-preformed-fibrils-pf-b39rqr56

and protocol for neuronal primary culture 
https://www.protocols.io/view/primary-culture-cortical-hippocampal-neurons-e15-1-cfiztkf6

 


[STEPS]
1. Aspirate the medium from coverslips (aspirate from only a few coverslips at a time).
2. Add 4% (wt/vol) PFA /4% (wt/vol) sucrose (1 ml for a 12-well plate).
3. Incubate the plate at Room temperature  for 15 min.

4. Wash 3 times 10 min with PBS/BSA/TWEEN solution.

5. Proceed or store in the fridge in PBS, protected from light, for up to 1 week.
6. Set up humidifier chambers.
7. Incubate with 0.5% Triton in PBS/BSA/TWEEN for 5 min. 

8. Wash 3 times 10 min  with PBS/BSA/TWEEN. 

9. Block in Donkey Serum 1:20 in PBS for 30 min.

10. During the blocking, prepare the primary antibody solution.
In PBS/BSA/TWEEN, add Biolegend psyn (MMS-5091) Mouse (1:1000) and Abcam MAP2 (ab5392) chicken Millipore (1:2000).
11. 
Incubate with primary antibody  for 60 min   at Room temperature. 

12. Wash 3 times 10 min  with PBS/BSA/TWEEN.

14. Incubate in secondary antibody solution for 60 min  at Room temperature  in the DARK.

15. Wash 3 times 10 min  with PBS/BSA/TWEEN.

16. Incubate with DAPI 1:10,000 in PBS 10 min  at Room temperature. 

17. Wash 3 times 10 min  with PBS.

18. Mount the coverslips onto glass slides with Prolong Gold mounting medium.

13. During the washes, prepare the secondary antibody solution:
In PBS/BSA/TWEEN, add Alexa Fluor 488 donkey anti-mouse (1:1000) + Alexa Fluor 594 donkey anti-chicken (1:1000).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>To amplify the target regions of GATA3 for studying rs3824662 polymorphism, a web-based primer designing tool available at <a href="http://bioinfo.ut.ee/primer3-0.4.0" target="_blank">http://bioinfo.ut.ee/primer3-0.4.0</a> facilitates crafting the set of outer primers [forward outer primer: 5’-TTGCAAATGGAAGAGGGTCT-3’ and reverse outer primer: 5’-ACCCTGCAAATGAGAGGAAA-3’] and inner primers [G specific primer: 5’-TGAGATTAAACACAAACACGtTG-3’ and T specific primer: 5’-CTGAGATTAAACACAAACACGaTT-3’] to perform allele-specific PCR that specifically amplify the GATA3 gene. G/T allele-specific primer is very sensitive and binds only with the DNA fragments containing G/T nucleotide template DNA. Forward outer and reverse outer primers will amplify a specific 691 base pair region of GATA3. This 691 base pair region contains the desired polymorphic site of interest. Allele-specific primers produce 506 bp products upon the presence of either a G allele or T allele. PCR reaction in a total volume of 15 μL with an initial denaturing step of 5 min at 95°C, followed by 40 cycles of 30 s at 95°C, 45 s at 55°C, and 1 min at 72°C, and a final extension step of 5 min at 72°C will produce better reproducible result. The reaction mixture contains both forward outer and reverse outer primers and contains either the G allele-specific forward inner primer or the T allele-specific forward inner primer. Primer concentration used was 200 nM each. When the G allele is present in a specific DNA sequence, only the 506 base pair band will be found upon gel electrophoresis of PCR amplicons with the G allele-specific primer. In this case, no such 506 base pair band will be obtained upon gel electrophoresis with the T allele-specific primer. On the other hand, for mutant TT genotypes, the opposite phenomenon will occur when the T allele is present, which will be reflected by the presence of the 506 base pair DNA band. In the case of the heterozygous genotype (GT), both inner primers for G and T alleles will bind with specific DNA sequences to produce 506 base pair bands.</p>

[BEFORE_START]
<p>Take precaution as required for a general PCR reaction.</p>

[STEPS]
?. 
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?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A collection of protocols designed to guide the user in processing a viral metagenome from raw sequence data to assembly, and subsequent analysis. The user uses <em>actual</em> reads from <a href="https://github.com/MicroB3-IS/osd-analysis" target="_blank">Ocean Sampling Day (2014)</a> and processes them entirely within Cyverse, a NSF-supported cyberinfrastructure.<br /><br />Quality trimming of reads is important!

[BEFORE_START]
To run this protocol, users must first <a href="https://user.iplantcollaborative.org/register/" target="_blank">register</a> for Cyverse account. All data (both inputs and outputs) are available within Cyverse's data store at /iplant/home/shared/iVirus/ExampleData/

[GUIDELINES]
This is part of a larger protocol <em>Collection</em> that involves the end-to-end processing of raw viral metagenomic reads obtained from a sequencing facility to assembly and analysis using Apps (i.e. tools) developed by iVirus and implemented within the Cyverse cyberinfrastructure.

[STEPS]
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TITLE: Quantification of area and optical density of intracellular neuromelanin with Image J
AUTHORS: Camille Guillard-Sirieix, Thais Cuadros, Miquel Vila

[DESCRIPTION]
Quantification of area and optical density of intracellular neuromelanin with Image J

[STEPS]
SECTION: Image Acquisition
1. Scan sections using 20x objective (NA=0.8) with pre-set focusing and exposure parameters for optimal NM signal quality with an automated Slide Scanner Olympus (SLIDEVIEW VS200, Tokyo, Japan).
SECTION: Image Acquisition
2. Acquire SNpc images with Qupath v0.5.0 software
SECTION: Neuromelanin Quantification
3. Upload images at Image J software
SECTION: Neuromelanin Quantification
4. Adjust canvas size at 1596x1198 
SECTION: Neuromelanin Quantification
5. Invert image
SECTION: Neuromelanin Quantification
6. With the free hand selections, draft a neuromelanin-pigmented neuron (excluding the nucleus) and measure the optical density (pixel brightness) and the cell area
SECTION: Neuromelanin Quantification
7. With the free hand selections, draft the neuromelanin pigment of the neuron and measure the optical density (pixel brightness) and the neuromelanin-occupied area
SECTION: Neuromelanin Quantification
8. With the free hand selections, draft 15-25 non-pigmented neurons (excluding the nucleus) and measure the optical density and calculate mean
SECTION: Neuromelanin Quantification
9. Normalize (i.e., subtract) the values of the neuromelanin pigmented neuron’s optical density with the mean value of the optical density of the non-pigmented neurons
SECTION: Neuromelanin Quantification
10. Additional: calculate the percentage of occupied area diving the neuromelanin pigment area by the neuron’s area