eamag/protocols-with-steps huggingface dataset:
TITLE: covid 19 indirect detection thru rise of 100nm filter fluid resistance
AUTHORS: Ladislav Nevery

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">By passing saliva sample thru 150nm filter to remove particles larger than avg 125nm sized covid 19 virus particles and concentrating resulting fluid to very small 1 mm2 area of 110nm filter. Pores in this small area should be clogged up fast raising filter air/fluid resistance. Due to high virus density 5.2 log10 ml saliva of infected person should clogg filter much faster than healthy person. Test is setup in such way that we let gravity pass 2l of fluid thru clogged filter and record rate of drops from its bottom as audio on mobile phone placed bellow glass cup. rate of drops thru clogged filter should be measurably slower than clean filter.</div></div>

[STEPS]
?. 3d print all required parts
?. place 25mm ideally 110nm pc or nylon membrane filter between blue parts. I used what I had piece
?. place 25mm ideally 150nm membrane filter in green saliva holder bellow red rubber gasket. I used what I had piece
?. .attach empty 2L soda bottle and squeze until all saliva passes thru both filters
?. remove green part  remove red parts and filter. clean green part
?. fill and reinstall bottle with 2l of clean fluid
?. place whole assembly  with bottle on top over any glass cup capable of holding blue part. place mobile phone  bellow glass cup and start audio recording to count and record sounds of drops.
?. stop audio recording. fill sample holder in step 4 with  fludid containin 120nm test nanoparticles in concentration resembling covid19  as5.2log10ml  and repeat whole process once more with new filters and clean parts
?. compare distance between drop sounds in all recordings.
drop frequency with 120nm nanoparticle clogged filter should be measurably different
TITLE: The Protocol of Adachi Rehabilitation Programme, Version1.0
AUTHORS: Yoshihiko Baba, Chika Ooyama, Yasushi Tazawa, Masahiro Kohzuki

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Objectives</span></div><div class = "text-block">We have developed Adachi Rehabilitation Programme (ARP), a community rehabilitation program in which older adults clean and plant flowers in the park and walk and shop in the shopping street under the supervision of professional caregivers. We examine the effects of ARP at small multifunctional at-home care.</div><div class = "text-block"><span style = "font-weight:bold;">Registration</span></div><div class = "text-block">The trial was registered at University Hospital Medical Information Network centre (UMIN000028317). The trial was also ethically reviewed and approved by Tohoku University Hospital Ethical Committee (2017-2-213 (Application-8996)) on 2 December 2017.</div></div>

[STEPS]
?. ARP is a weekly exercise and occupational therapy that facilitated participation with low (In the first week, participants visit a shopping street to purchase the items for park cleaning and gardening. The total duration of transportation and shopping is approximately three hours. In weeks two to four, participants go to a nearby park for cleaning and gardening with or without walking aids. For cleaning, participants hold a pair of tongs or a bin liner and walk around in the park to pick up litters. For gardening, they dig the ground, plant flowers and water them. The supervised intervention is carried out once a week, but the participants are encouraged to visit the park to take care of the flowers on other days. This four-week series is repeated three times.The frequency, intensity, type and time of physical activities are summarized in Table 1.Table 1. The basic components of Adachi Rehabilitation Programme  ABCD1FrequencyIntensityTypeTime2Once a weeklow to moderateWalk to bus stop103(Week 1)lowBus ride304lowStroll in the shopping street605Rest106lowBus ride307low to moderateWalk from bus stop108Total150910Once a weeklow to moderateWalk to park511(Weeks 2 to 4)moderatePlant flowers1012lowPick up garbages4013low to moderateWalk from park514Total60Note: The unit of time is minutes.The basic component is a four-week programme. The basic component was repeated three times (12 weeks).
ABCD1FrequencyIntensityTypeTime2Once a weeklow to moderateWalk to bus stop103(Week 1)lowBus ride304lowStroll in the shopping street605Rest106lowBus ride307low to moderateWalk from bus stop108Total150910Once a weeklow to moderateWalk to park511(Weeks 2 to 4)moderatePlant flowers1012lowPick up garbages4013low to moderateWalk from park514Total60
TITLE: (SCAM LEGIT)Tamra Judge CBD Gummies Reviews, Benefits, Price, Offers, Where To Buy?
AUTHORS: Tamra Judge CBD Gummies

[DESCRIPTION]
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[STEPS]
TITLE: 1M KPO4 pH6.0
AUTHORS: Ida Barlow

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for making phosphate salt solution for adding to NGM</div></div>

[STEPS]
?. [Make 1M solutions of monobasic and dibasic salts]
Prepare 1M solution dibasic salt: K2HPO4 water
174.18 g
1 L
?. [Make 1M solutions of monobasic and dibasic salts]
Prepare 1M solutions of monobasic salt:  KH2PO4 water
136.09 g
1 L
?. [Make 1M solutions of monobasic and dibasic salts]
Autoclave both solutions and allow to cool
?. [Mix salts to achieve correct pH]
Mix prepared salt solutions to achieve 1M KPO4 solution close to pH6: 1M K2HPO4 (dibasic) 1M KH2PO4 (monobasic)Adjust pH to phH6.0 using pH meter and addition of drops of KOH
100 mL
400 mL
TITLE: Preparation of Free Floating Coronal Mouse Brain Sections
AUTHORS: madalynn.erb Erb

[DESCRIPTION]
Protocol to prepare free floating mouse brain sections for immunostaining or in situ hybridization.

[STEPS]
SECTION: Collect Mouse Brain Tissue
1. Deeply anesthetize mice via intraperitoneal injection of 2X Avertin solution
SECTION: Collect Mouse Brain Tissue
2. Perform transcardial perfusion using chilled saline solution
SECTION: Collect Mouse Brain Tissue
3. Switch from saline solution to chilled PFA
SECTION: Collect Mouse Brain Tissue
4. Remove brain immediately after PFA perfusion
SECTION: Collect Mouse Brain Tissue
5. Incubate brain in PFA for 24 hours at 4°C
SECTION: Collect Mouse Brain Tissue
6. Transfer brains to 30% Sucrose / 0.1M Phosphate Buffer (PB) – keep at 4°C for ≥ 24 hours
SECTION: Collect Mouse Brain Tissue
7. Tissue should be completely saturated with sucrose before sectioning
   
SECTION: Section Tissue
8. Use a Leica SM2010R Microtome
SECTION: Section Tissue
10. Chill the microtome platform by covering it with crushed dry ice
SECTION: Section Tissue
15. Gently cover the brain in crushed dry ice to freeze the tissue
SECTION: Section Tissue
18. Seal plate with parafilm and store tissue stored at -20°C
SECTION: Section Tissue
8.1. Blade: Leica 16cm, knife angle set at 0 degree 
SECTION: Collect Mouse Brain Tissue
2.1. 0.9% NaCl kept on ice
SECTION: Collect Mouse Brain Tissue
2.2. Use approximately 60mL saline solution per mouse
SECTION: Collect Mouse Brain Tissue
3.1. 4% paraformaldehyde in 0.1M phosphate buffer (PB) pH 7.4

SECTION: Collect Mouse Brain Tissue
3.2.  Use approximately 60mL PFA per mouse
SECTION: Collect Mouse Brain Tissue
7.1. Brains will sink to the bottom of the vial when saturated
SECTION: Section Tissue
8.2. Cut thickness: 35µm
SECTION: Section Tissue
11. Apply 5-10 drops of 30% Sucrose / 0.1M Phosphate Buffer (PB) solution to the chilled microtome platform and wait for it to solidify
SECTION: Section Tissue
12. Use the microtome to gently shave the solidified sucrose to make a flat surface
SECTION: Section Tissue
9. Adjust the microtome platform so that it is level with the blade and lock it into place
SECTION: Section Tissue
13. Use a razor blade to remove any spinal cord from the brain making a flat surface perpendicular to the rostral / caudal axis
SECTION: Section Tissue
14. Apply 2-3 drops of sucrose to the existing sucrose platform and quickly position the brain with the olfactory bulbs pointing upwards
SECTION: Section Tissue
14.1. Apply 2-3 more drops of sucrose to the top of the brain to securely freeze it to the microtome platform
SECTION: Section Tissue
16. Adjust the microtome platform so that the rostral / caudal axis is perpendicular to the blade and the dorsal / ventral axis is level with the blade
SECTION: Section Tissue
17. Collect sections in a 24-well plate prefilled with cryoprotectant solution (0.1M PB +30% Sucrose + 30% Ethylene Glycol)
SECTION: Recipes
19. 0.2M Phosphate Buffer (PB)
 
 
 
SECTION: Recipes
20. 4% PFA
 
 
 
SECTION: Recipes
21. Saline
 
 
 
SECTION: Recipes
22. 30% Sucrose
 
 
 
SECTION: Recipes
23. Cryoprotectant Solution
TITLE: TCA protein extraction from diatoms
AUTHORS: Anna Santin, Antonella Ruggiero, Francesco Manfellotto, Mariella Ferrante

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protein extraction from diatoms using TCA</div></div>

[STEPS]
?. Pellet of around 50 ml of culture in exponential phase (2 million of cells)
?. Wash pellet with 1 ml of TCA 20%
?. Centrifuge at maximum speed for 1 minute
?. Pour off the supernatant and dilute in 100 µl of TCA 20%
?. Add Glass-Beads till meniscus and vortex for 7 minutes
?. Add 400 µl of TCA 5% and transfer the supernatant in new eppendorf (1.5 ml)
?. Centrifuge at 3000 rpm for 10 minutes (chiarification phase)
?. (now quickly!)Add 100 µl of 3X SDS Laemmli Buffer (see Materials) and vortex very well to dissolve
?. Add 50 µl Tris Base and vortex very well to dissolve
?. Boil at 100°C for 3 minutes
?. Centrifuge at maximum speed for 5 minutes
?. Transfer supernatant containing proteins in a new eppendorf
?. Put tubes on ice
?. Stock at -20°C
?. Pour off supernatant by aspiration
TITLE: 10x Genomics Xenium for FFPE User Guides
AUTHORS: Andrew Houston

[DESCRIPTION]
Xenium allows for in situ transcript detection of selected probe set of RNA targets revealing spatially resolved expression patterns at nearly single-cell resolution. 

[STEPS]
SECTION: Tissue processing and QC
1. Determine suitable samples of interest by performing a test described in CG000578, sectioning guidelines described in Step 1 pgs. 18-25 and H&E staining in Appendix pgs. 38-45
  

SECTION: Tissue processing and QC
2. Evaluate section and staining quality, only choose samples or regions free of detachment or artifacts 
SECTION: Xenium slide preparation
3. Utilize CG000578 for section placement on Xenium slides, follow Step 3, pgs. 29-33
SECTION: Xenium analyzer
6. Prepare Xenium instrument as outlined in CG000584  

SECTION: Xenium slide preparation
4. After drying slide overnight or up to 4 weeks, proceed with deparaffinization and decrosslinking steps found in CG000580   

SECTION: Xenium slide preparation
5. Immediately following decrosslinking start to prepare probes of interest as outlined in CG000582   
Initiate probe hybridization overnight, continue with the protocol the next day

SECTION: Xenium analyzer
7. Load slides and initiate sample scan
TITLE: Standard operating procedure for the isolation of genetically engineered hPSCs clones in a high-throughput way
AUTHORS: Hanqin  Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This collection describes a standard procedure for isolating single human pluripotent stem cell (hPSC) clones in a high-throughput way. This collection follows nucleofection of hPSCs as described in detail in the collection "Nucleofection (Amaxa) and electroporation (Biorad) of hPSCs;" dx.doi.org/10.17504/protocols.io.b4qnqvve

Collection overview
Seeding nucleofected hPSCs in 96-well plates using limited dilution
Duplicating 96-well plate-cultured hPSCs clones 
Subcloning of genotype-confirmed hPSCs clones

General notes
1. Throughout these protocols, the term hPSC is used to collectively refer to both hiPSCs and hESCs. All described procedures have been tested and work equally well for hiPSCs and hESCs.



[STEPS]
TITLE: PARENTING AND SELF CRITICISM AMONG OFFSPRING: A SYSTEMATIC REVIEW
AUTHORS: Mafia Shahzadi

[DESCRIPTION]
Background:Self-criticism is the drill of negative self-appraisal and a tendency in which the individual reacts critically towards the self and points out one's own beliefs, actions, and thoughts that make them more dysfunctional. Researches supported that parenting is one of the perspectives which make an individual self-critical. Parents as primary caregivers significantly influence children's lifestyles. Such as humble, well-being, distress, and self-criticism. We conducted a systematic review to evaluate the relationship between parenting and self-criticism.
Methods:Scopus, Web of Science, Google Scholar, MEDLINE, Science Direct, PsycINFO, PubMed, and OvidSP databases were searched that consisting literature published around 2003 to 2019. The researcher explored the data between June to September 2021.
Result:Atotal of 40 articles were identified from electronic databases. Based on abstracts
and titles, ten papers were excluded from this review. Furthermore, 25 articles were excluded after assessing full text for not meeting inclusion criteria, and we included 11 relevant articles.
Conclusion:It is concluded that almost all studies revealed a strong relationship between parenting and self-criticism. Parenting affects the level of self-criticism as well as parental
rejection is a more significant factor in intense Self-criticism.

Keywords: 

[BEFORE_START]
Before June 2021

[GUIDELINES]
PARENTING AND SELF CRITICISM AMONG OFFSPRING: A SYSTEMATIC REVIEW

[STEPS]
SECTION: PARENTING AND SELF CRITICISM AMONG OFFSPRING: A SYSTEMATIC REVIEW
1.
TITLE: 30% Sucrose for Cryoprotection of brains after Perfusions
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare 30% Sucrose Solution in 0.1M phosphate buffer. 30% Sucrose in 0.1M phosphate buffer is used for the cryoprotection of mice brains post perfusion.</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: Splitting Adherent Cell Lines
AUTHORS: Carolina Lopez

[DESCRIPTION]
This protocol describes how to split and maintain adherent cell lines in culture. Examples of these cells are: A549 cells, LLCMK2 cells, and MDCK cells.

[STEPS]
SECTION: Protocol
1. Splitting Adherent cells
Wash flask 2x with sterile PBS.
Add 2mL of trypsin/T75. Incubate at 37ºC for 2-3 mins or until cells are detached from the flask.
Add 8mL of cell culture media and pipette up and down. Transfer all the media to a 15mL tube. 
Centrifuge at 1200 rpm for 5 min. 
Discard media and resuspend cells in 10mL Tissue Culture Medium
Add 2mL of cell suspension to T75 flask, then add 8mL Tissue Culture Medium. The cell will be ready for next split two days later.
TITLE: Detection of Tau ubiquitylation
AUTHORS: Itika Saha, F. Ulrich Hartl, Mark S. Hipp

[DESCRIPTION]
This protocol describes the detection of ubiquitylated Tau from HEK293 cells stably expressing and propagating aggregates of Tau repeat domain fused to YFP (Sanders et al. Neuron, 2014; Saha et al, BioRxiv, 2022).

[STEPS]
1. Harvest cells from 2 confluent wells of a 6 well plate.
2. Lyse aggregate-containing cells by vortexing in cold RIPA buffer (Thermo) supplemented with protease inhibitor cocktail (Roche) and DNase. 20 mM N-ethylmalemide should be included in the lysis buffer to inhibit the activity of deubiquitinating enzymes.
3. Briefly sonicate lysate and centrifuge at 2,000 x g for 5 min to remove cell debris.
4. Collect supernatant, determine protein concentration and normalize across samples.
5. Dilute 1 mg protein in a total volume of 600 µL RIPA buffer.
6. Add 50 µL anti-GFP bead slurry (µMACS GFP Isolation kit, Miltenyi Biotec) to diluted lysate.
7. Incubate for 1 h at 4 °C in a rotating wheel at 10 rpm.
8. Before the end of 1 h, place µ-columns (Miltenyi Biotec) in the magnetic field of a µMACS Separator (Miltenyi Biotec) and equilibrate columns by applying 250 µL RIPA buffer. Allow complete flow-through.
9. Apply cell lysates and beads to µ-columns. Allow complete flow-through.
10. Wash columns 4 times with 1 mL 0.1% SDS/PBS.
11. Elute by applying 50 µL pre-heated (95 °C) 1x SDS sample buffer.
12. Analyze eluates by immunoblotting with antibodies against GFP or ubiquitin.
TITLE: Preparation of horseradish peroxidate (HRP) conjugated Peptostreptococcal protein-L  by the periodate method.
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This reagent can be used in ELISA, Western blotting and Dot blot to detect antigens and antibodies. It is important in the immunodiagnosis of infectious diseases and other problems. Protein L binds to kappa light chains of immunoglobulins from many animal species including human, mouse, rat, chicken, hamster and pig [1].</div><div class = "text-block"><span>1. Justiz-Vaillant AA, Akpaka PE, McFarlane-Anderson N, Smikle MF. Comparison of techniques of detecting immunoglobulin-binding protein reactivity to immunoglobulin produced by different avian and mammalian species.</span><span style = "font-style:italic;">West Indian Med J</span><span>. 2013;62(1):12-20.</span></div></div>

[STEPS]
?. Horseradish peroxidase (500 µg in 50 µl NaCO3 , pH 9.6) is mixed with freshly made sodium periodate solution (1.71 mg/ml) followed by incubation in the dark for 2 h.
?. Mix 500 µg of protein-L (SpL) with an equal amount (500 micrograms) of a mix of horseradish peroxidase-sodium periodate.
?. The mixture is incubated for 3 hours at 4°C with gentle agitation.
?. Forty µl of freshly prepared NaBH4 solution (5 mg NaBH4 /ml 0.1 mM NaOH) is then added to the preparation.
?. The preparation is incubated for 90 min at 4°C in the dark with gentle agitation.
?. Cold 50% saturated ammonium sulphate solution (pH 7.4) is added drop by drop in the ratio 1:1 (v/v).
?. The mixture is then centrifuged for 25 min at 4°C and recover the pellet at the bottom of the tube.
?. The pellets is re-suspended in 200 µl of PBS pH=7.4 and dialysed against 1L of PBS for 24 h with 3 buffer changes.
?. An equal volume of glycerol is added to the dialysate followed by 100 µl of bovine serum albumin, BSA (20 mg/ ml).
?. The conjugate is then stored at -20°C until further used.
TITLE: ASAP protocol: Crystallization of Enterovirus D68 3C protease (PDB code 8CNX)
AUTHORS: ryan.lithgo, Peter Marples, daren.fearon, Lizbé Koekemoer

[DESCRIPTION]
The crystallization protocol and buffer conditions used to obtain Enterovirus D68 3C protease crystals suitable for XChem fragment screening. The crystal structure was depostited to RCSB PDB with code 8CNX.

[STEPS]
SECTION: Crystallization experiment
2. Protein and buffer requirements:
32 µL1 millimolar (mM)  
2.88 mL 
16 µL   seeds, dilution 1:100 000

SECTION: Crystallization experiment
5. Dispense 30 µL into SwissCI 3 lens plate reservoir wells using a 100 µl multi-channel pipette.
Dispense 100 nL1 millimolar (mM)    to each lens using the SPT mosquito.
Dispense  200 nL to each lens using the SPT mosquito.
Dispense  50 nL to each lens using the SPT mosquito.

Drop ratio: 1:2:05 ratio (100 nl   : 200 nl reservoir solution: 50 nl seeds)
Final drop volume: 350 nl
SECTION: Crystallization experiment
3. Crystallisation screen composition:
0.1 M Tris-HCl (pH 8.14)
0.2 M Ammonium acetate
25% w/v PEG 3350

Stock solutions used:
1 M Tris-HCl adjusted to pH 8.14 with HCl
1 M Ammonium acetate
50% w/v PEG 3350

 

SECTION: Crystallization experiment
7. Crystal form after ~12 h.
 


SECTION: Crystallization experiment
6. Incubate at 20 °C for 1440 min in Formulatrix Rock Imager.

Imaging Schedule: The first images are taken after 12 h and the imaging schedule follows a Fibonacci sequence of days for further collections. 
SECTION: Equipment needed
1. Formulatrix Rock Imager (or incubator of choice)
SPT mosquito
P100 multi-channel pipette
SwissCI 3 lens plate

SECTION: Crystallization experiment
4. Prepare seed stock:
1: 100 000 dilution    seeds
TITLE: Plasmid construction
AUTHORS: Adam  Yokom

[DESCRIPTION]
plasmid construction

[STEPS]
5. Purify the DNA from gel using a Gel extraction kit (Bio Basic).
2. Linearize the backbone with restriction enzymes (NEB). 
1. Amplify the insert gene fragment by PCR with primers including 21 nt of overlapping sequence with the target gene.  
4. Run  PCR products and linearized backbone in an agarose gel to confirm the size. 
6. Ligate the linearized backbone and the insert with the T4 ligase (NEB). 
3. Treat linearized backbone with quick CIP (NEB). 
7. Transform the ligation products into home made competent cells. 
8. Perform colony PCR to screen for colons that with inserted gene. 
9. Sequence to verify that the inserted gene is correct.
TITLE: Injection of cholera toxin subunit B in the interscapular brown adipose fat and Stellate Ganglion dissection
AUTHORS: Seoeun  Lee, Daniele  Neri, Lori  Zeltser

[DESCRIPTION]
Protocol to deliver cholera toxin subunit B into the interscapular brown fat of mice

[STEPS]
SECTION: Preparation
2. Anesthetize the animal under isoflurane until there's no toe pinch reflex
SECTION: Preparation
1. Prepare the surgery area by placing a clean paper towel on top of a heat pad
SECTION: Preparation
3. Transfer the mouse to the surgery area, flat on its stomach on the heatpad. Place the nose cone to maintain anesthesia
SECTION: Preparation
4. Shave the interscapular area of the mouse and sterilize it with three alternating wipes of betadine and 70% isopropyl alcohol
SECTION: Preparation
5. Apply eye ointment
SECTION: Preparation
6. Administer analgesics. We use buprenorphine 0.1 mg/kg subcutaneously
SECTION: Surgery
8. Make a 1 cm horizontal incision above the interscapular area 
SECTION: Surgery
9. Visually identify the white adipose layer of interscapular pad, and use scissors to cut it and expose the underlying brown adipose tissue
SECTION: Surgery
10. While holding the tissue to expose the brown fat, inject 10 uL of CTb on each side
SECTION: Preparation
7. Load up cholera toxin subunit B (CTB) in a glass pulled needle connected to a mouth pipette (10 uL per side)
SECTION: Surgery
11. Gently return the fat pads to their original position, and close the wound with a 4-0 PS-4 suture 
SECTION: Surgery
12. Clean the wound with betadine and 70% isopropyl alcohol and monitor the animal for any sign of distress
SECTION: Tissue harvesting
13. Euthanize the mouse after 72 hours by intraperitoneal injection of 2-2-2-Tribromoethanol (500 mg/kg)
SECTION: Tissue harvesting
14. Open the ribcage and remove the lungs for easier access to the sympathetic chain
SECTION: Tissue harvesting
15. Carefully dissect the Stellate Ganglion from each side
TITLE: Faktorová_et_al_2019_pTpPuc3_TpSil3p-eGFP
AUTHORS: Jernej Turnsek

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Details of the conjugation episome used to generate the TpSil3p-EGFP expressing </span><span style = "font-style:italic;">Thalassiosira pseudonana</span><span> strain as reported in Faktorová et al (2019). The file contains:</span></div><div class = "text-block">1/ a vector scheme summarizing all of its elements</div><div class = "text-block">2/ expression cassette elements and their nucleotide sequences</div><div class = "text-block">3/ fusion protein amino acid sequence</div><div class = "text-block">4/ full vector DNA sequence with expression cassette elements annotated</div></div>

[STEPS]