eamag/protocols-with-steps huggingface dataset:
TITLE: Immunohistochemistry of rTg4510 mouse brain
AUTHORS: Miguel Da Silva Padilha, Irina Dudanova, Itika Saha, F. Ulrich Hartl, Mark S. Hipp

[DESCRIPTION]
This protocol describes immunohistochemical staining of the Tau transgenic mice rTg4510 (Santacruz et al, 2005) brain section for Tau aggregates phosphorylated at Ser202/Thr205 and the AAA+ ATPase Valosin-containing protein (VCP). Experiments involving animal models must be performed in accordance with relevant institutional guidelines and regulations. 

[STEPS]
1. Deeply anesthetize mice with 1.6% Ketamine/0.08% Xylazine and transcardially perfuse with PBS followed by 4% paraformaldehyde (PFA)(Santa Cruz) in PBS.
2. Dissect brains out of the skull and post-fix in 4% PFA in PBS overnight.
3. Embed fixed tissue in agarose and section into 40 μm thick sections using a vibratome (VT1000S, Leica). 
 

4. Permeabilize sections with 0.5% Triton X-100 and wash with PBS.
5. Incubate sections in blocking solution consisting of 0.2% BSA (w/v), 5% donkey serum (v/v) (abcam), 0.2% lysine (w/v) (Sigma-Aldrich), 0.2% glycine (w/v) (Sigma-Aldrich) in PBS for 30 min at room temperature (RT).
6. Incubate sections with primary antibodies (anti-phospho-Tau AT-8 (Thermo, Cat# MN1020, 1:300); anti-VCP (Novus Biologicals, Cat# NB 100-1558, 1:500) diluted in 0.3% Triton X-100 (v/v), 2% BSA (w/v) in PBS overnight at 4°C.
7. Wash sections in PBS and incubate with secondary antibodies and Neurotrace 640/660 (ThermoFisher, Cat# N21483, 1:500) diluted in 0.3% Triton X-100, 3% donkey serum (v/v) for 2 h at RT.
8. Stain nuclei with 0.5 µg/ml DAPI.
9. Mount sections on Menzer glass slides using Prolong Glass fluorescence (Invitrogen) mounting medium.
TITLE: S2: Step-by-step-guide using MeshLab in the work flow.
AUTHORS: Paul Kalke, Conrad Helm

[DESCRIPTION]
How to do data refinements like repair, smooth and simplification in MeshLab and export as -ply-file.

[STEPS]
1.   
•Open MeshLab
•First step is Cleaning and Repairing your mesh-irrelevant if it is generated by Image J, Amira or any other software, the steps are the same
•Often used procedures/steps:
-Remove Duplicate Faces
-Remove Dublicate Vertices
-Merge Close Vertices
-Repair non Manifold edges by…
•Just try out, the results are written in the downright white window

--> Also see: more specific
https://www.youtube.com/watch?v=aoDLrXp1sfY
https://www.youtube.com/watch?v=j9EKk3Bs1TQ
https://www.youtube.com/watch?v=gWBm5XGRJOk



--> more general
Mister P. MeshLab Tutorials
https://www.youtube.com/playlist?list=PL8B1E816EAE236B4D
2.   
•Now re-mesh your label by Remeshing: Isotropic Remeshing
•You can do that multiple times but 1 or 2 is enough to change the topology of your vertices in a more regular distribution
3.   
•Result of re-meshing shown in Blender
4.   
•A smoothing step is possible here but not obligatory
•Taubin smooth or Laplacian Smooth are suitable and can be used also multiple times
5.   
•Result of 2 times Taubin Smooth shown in Blender
6.   
•Now you reduce the size by decreasing the vertices of your label/surface by Simplification: Quadric Edge Collapse Decimation
7.   
•Result of 2 times simplification shown in Blender
8.   
•Save label as .ply-file
•Repeat all described steps for each label or batch of labels by opening a new file
TITLE: Dose dependent test
AUTHORS: Hung Liang Pai, Cheng-Ruei Yang

[STEPS]
?. [Preparation]
Sterilize the bench, and put on a labmat
?. [Preparation]
Thaw the reagents including 1. DNase/ RNase free water (store in -20°C) 2. solution A (store in -80°C) 3. solution B (store in -80°C) 4. RNase inhibitor (store in -20°C) 5.toehold switches with invertase DNA (store in 4°C) 6.miRNA as the trigger (store in 4°C)
on ice
?. [Prtotocols]
Gently shake and spin down the tube before adding (especially solution A and DNA plasmid)
?. [Prtotocols]
Add below reagents in to PCR tubes in order:1. ddH2O(till totally )2. solution A3. solution B4. inhibitor5. toehold switches with invertase DNA6. miRNA
5 µl
2 µl
1.5 µl
0.2 µl
50 ng
0 µl
50 µl
100 µl
150 µl
200 µl
?. [Prtotocols]
Centrifuge
Centrifuge: 4000 33
?. [Prtotocols]
Place the PCR tubes in the dry bath incubator
37 °C
?. [Prtotocols]
Put the PCR tubes   after 2 hours
on ice
?. [Prtotocols]
Measure the glucose concentrations of each PCR tubes by glucometer
?. [Prtotocols]
Put the PCR tubes in the dry bath incubator
55 °C
?. [Prtotocols]
Measure the glucose concentrations of each PCR tubes by glucometer again
TITLE: 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: Linguistic signaling, emojis, and skin tone in trust games
AUTHORS: J Jobu Babin

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This paper reports the results of an experiment involving text-messaging and emojis in laboratory trust games executed on mobile devices. Decomposing chat logs, I find that trust increases dramatically with the introduction of emojis to one-shot games, while reciprocation increases only modestly. Skin tones embeddedin emojis impact sharing and resulting gains – to the benefit of some anddetriment to others. Both light and dark skin players trust less on receipt of a dark skin tone emoji – suggestive of statistical discrimination. In this way, computer-mediated communication leads to reduced gains for dark-skinned persons. These results highlight the complex social judgment that motivates trust in an anonymous counterpart.</div></div>

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Protocol established by Anna F. Zetler and published by Luisa F. Jiménez-Soto</p>
<p>This protocol is the final adaptation of protocols used in the laboratory of Prof. Rainer haas. The following people contributed with their ideas and experience: Benjamin Busch, Bettina Vogl-Gebert and Luisa F. Jiménez-Soto</p>
<p> </p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Heat Inactivate FBS
AUTHORS: Cody Roberts

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Heat inactivate FBS to denature compliment. Critical for trypanosomes but not for EA.hy 926 cells. </div></div>

[STEPS]
?. Allow serum to acclimate at room temperature for a minimum of 10 minutes or refrigerate overnight at 2° C to 8° C.
?. The serum may then be completely thawed at room temperature.  Or, you may choose to proceed by thawing the serum in a water bath at 37° C.  Thawing serum at temperatures above 37° C or for an extended period of time could lessen the quality of serum and possibly cause increased amounts of precipitates and/or cause a cloudy appearance to the serum.  Swirl the bottles throughout the thawing process every 10-15 minutes as this will disperse the released salts and proteins uniformly.
?. Prepare a water bath, if not already done so during the thawing process, that can be temperature controlled to 56° C for the heat inactivation process.  There must be enough water to submerge the entire contents of all bottles.
?. Fill a control bottle with water to monitor the water bath temperature.  The control bottle must be identical to the bottles being heat inactivated and the temperature of the water within the control bottle should be the same as that of the thawed serum.
?. Place the control bottle and serum into the 56° C water bath.  Suspend a calibrated thermometer into the control bottle to monitor the temperature during the heating process.  Do not allow the thermometer to touch the sides or bottom of the bottle.
?. Set a timer to 30 minutes once the temperature of the control bottle has reached  56° C.  Maintain this same temperature the entire duration of the process. Gently swirl the bottles every 3-5 minutes to make certain the serum remains uniform throughout heating.
?. Remove the heat inactivated serum after 30 minutes and gently swirl once again.
?. In cell culture hood, aliquot 50 mls into 10 50 ml containers.
?. Store the heat inactivated serum at -20° C in frosted freezer.  Caution to Note:  The following possibilities may cause protein precipitates:
?. Caution to Note:  The following possibilities may cause protein precipitates:1.) Improper Thawing 2.) Inactivating serum that has not been thoroughly mixed homogeneously 3.) Inactivating serum above the recommended temperature 4.) Inactivating serum for a longer period of time than recommended 30 minutes
?. Label as Heat Inactivated FBS
TITLE: Passive avoidance (step-down test)
AUTHORS: Marina Lorente Picón, Núria Peñuelas, Ariadna Laguna, Miquel Vila

[DESCRIPTION]
Passive avoidance (step-down test) for mice

[STEPS]
1. Prepare the platform: Passive Avoidance - Step Down for Mice (vibrating platform) (#40570, Ugo Basile) consisting of a platform located in a controllable electrified net. 
2. Train the animals 24h before the test. Put the animal in the plexiglass platform located in the middle of the electrified net with 0.3 mA. If the mouse steps down the platform, it will receive an electric shock. Stop immediately the electrified net and remove the animal from the cage. 
3. Clean the platform each time between animals.
4. After 24 hours, place again the same mice in the same platform. Do not electrify the net. Measure the time that the animal spends on the platform before stepping down.
5. Calculate the time on the platform by subtracting the latency time from the first day to that of the second day to normalize for the inter-individual variability in curiosity/activity of each mouse.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Drop-seq is a technology we developed to enable biologists to analyze RNA expression genome-wide in thousands of individual cells at once.  We first described this in <a href="http://www.sciencedirect.com/science/article/pii/S0092867415005498" target="_blank">a 2015 paper in <em>Cell</em></a>.  Though commercial implementations of droplet-based single-cell RNA-seq also now exist, we have made Drop-seq open-source and want to make sure that any lab can build their own system.  The materials for constructing a Drop-seq setup in one’s own lab can be obtained for about $6,000. The reagents for performing Drop-seq cost about 6 cents per cell.</p>
<p> </p>
<p>This is a supplemental protocol of our <a href="https://www.protocols.io/view/drop-seq-laboratory-protocol-mkbc4sn" target="_blank">Drop-Seq Protocol</a> for measuring droplet volume in home-made microfluidic devices. </p>

[BEFORE_START]
<p style="text-align: justify;">To measure droplet volume, purchase some durable, monodisperse polystyrene beads with a hydrophilic coating (e.x. 10­‐micron carboxylated polystyrene beads from Bangs Labs, product #PC06N-11355. It can be helpful to use fluorescent beads to be sure you can identify them in droplets. Bangs cells these under product # FC06F‐10163).</p>
<p> </p>

[GUIDELINES]
<p> <strong>Videos and FAQs </strong></p>
<p> </p>
<p>These <a href="http://mccarrolllab.com/drop-seq-troubleshootingtutorial-with-pictures-videos/" target="_blank">tutorials, images, and diagrams</a> may be helpful in building your own Drop-seq setup and doing Drop-seq experiments in your lab.</p>
<p> </p>
<p>We have also created a YouTube channel with a variety of <a href="https://www.youtube.com/channel/UCRptwggZzFyM51R5iAI-07Q" target="_blank">videos</a> to help scientists through the steps that most benefit from watching.</p>
<p> </p>
<p>This <a href="http://mccarrolllab.com/drop-seq-faq/" target="_blank">FAQ</a> provides also provides supplementary information.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Isolation of ECs from Lymph node tissue for scRNAseq
AUTHORS: Guido  Krähenbühl

[DESCRIPTION]
Protocols describing the isolation of murine lymph node endothelial cells
Isolation of lymphatic endothelial cells for spectral flow cytometry and scRNA-seq
Basic analysis of spectral flow cytometry data of endothelial cells and scRNA-seq 
Adapted from https://doi.org/10.1016/j.xpro.2022.101267

[STEPS]
SECTION: Preparation
1. Day before experiment check Antibodies to be used and prepare the following Reagents:


 

 

 

SECTION: Tissue collection
3. Sacrifice mice using Isofluorine 
(do not use cervical dislocation as this might affect perfusion of the brain)
SECTION: Tissue collection
4. Dissect inguinal, axillary, brachial and cervical lymphnodes

  

SECTION: Tissue collection
5. Perform transcardial perfusion via the left ventricle with 8 mL  ice-cold
 at a perfusion rate of 2 mL/min for 5 min.
SECTION: Tissue collection
6. Dissect cervical lymph nodes and collect in 5 mL 

SECTION: Tissue collection
7. Collect remaining lymphnodes (axillary, brachial and inguinal) and add the to the cervical lymph nodes
SECTION: Tissue collection
8.   



SECTION: Tissue processing
9. Remove the RPMI-1640 and add 5 mL  at 37 °C 15 min 

SECTION: Tissue processing
10. Aspirate and pipette the solution 10 times using a 5 mL serological pipette
SECTION: Tissue processing
11. Allow large fragments to settle down and transfer supernatant to 20 mL , add fresh 5 mL  to remaining fragments and incubate at 37 °C 15 min 
repeat 4 times or until fragments are fully digested
   

SECTION: Tissue processing
12. Centrifuge pooled supernatants at 300 x g, 5 min, 4 °C 

SECTION: Tissue processing
13. Resuspend in 5 mL

SECTION: Preparation
2. Before starting prepare:
Dissection Tools
If Brain tissue is also taken, prepare perfusion kit
5mL RPMI-1640
Keep all reagents on ice
SECTION: FACS Sort
17. Incubation with blocking antibody for 10 min at 4°C (CD16/CD32, 1:100 in FACS buffer)
SECTION: FACS Sort
18. 
Add Antibodies, mix and incubate at 4 °C 30 min 

 

SECTION: Tissue processing
14. Centrifuge at 300 x g, 5 min, 4 °C 

SECTION: Tissue processing
15. Resuspent in 500 µL 

SECTION: FACS Sort
19. Wash cells twice with FACS buffer by centrifugation at 300 rcf, 5 min, 4 °C  and resuspend in 300 µL 

SECTION: eryhtrocite lysis
16. Lyse erythrocytes with lysis buffer 
(can result in loss of sensitive endothelial cells)
SECTION: FACS Sort
20. add viability dye immediately befor analysis (7-AAD at 1:100, DAPI at 1:100)
SECTION: FACS Sort
21. Gating and sorting
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Zobell plates, 100% nutrient (1 L)</strong><br />26 g sea salts<br />1 g yeast extract<br />5 g proteose peptone no. 2<br />fill to 1 L qH<sub>2</sub>O<br />12 g agar<br /><br />Autoclave 1 L PZM for 50 min.  Pour 10 ml/plate.<br /><br />Grow Pseudoalteromonas in 50% nutrient PZM (Pseudoalteromonas Zobell Medium).  For 50% nutrient PZM, reduce the amount of Proteose Peptone No. 2 in the above recipe to 2.5 g per 1L. <br /><br /><strong>Zobell plates, 20% nutrient (1 L)</strong><br />26 g sea salts<br />0.2 g yeast extract<br />1 g proteose peptone no. 2<br />fill to 1 L qH<sub>2</sub>O<br />12 g agar<br /><br />The 20% nutrient Zobell is used for plaque assay bottom agar.<br /><br /><strong>Zobell liquid, (1 L)</strong><br />26 g sea salts<br />1 g yeast extract<br />5 g proteose peptone no. 2<br />fill to 1 L qH<sub>2</sub>O<br /><br /><strong>Top Zobell agar (1 L)</strong><br />26 g sea salts<br />1 g yeast extract<br />5 g proteose peptone no. 2<br />fill to 1 L qH<sub>2</sub>O<br />6 g agar<br /><br />Top agar is semi-solid (uses half the amount of agar as normal bottom agar).<br /><br /><strong>MSM (1 L)</strong><br />100 mM NaCl (5.8 g)<br />81.2 mM MgSO<sub>4</sub>·7H<sub>2</sub>O (20 g)<br />50 mM Tris base (3.02 g) OR 50 mL 1 M Tris (pH 7.6)<br />fill to 1 L qH<sub>2</sub>O<br />adjust to pH 7–8 if necessary<br /><br /><strong>Artificial seawater, 26 psu (ASW; 1 L)</strong><br />26 g sea salts<br />fill to 1 L qH<sub>2</sub>O<br /><br />

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol provides a procedure for mapping reads to co-assembled contigs using Bowtie2. </p>

[BEFORE_START]
<p>If you haven't already, you should consolidate your fasta files into their own directory (seperate from the fastq files). From /rsgrps/bh_class/username : </p>
<p> </p>
<p>$ mkdir fasta</p>
<p> </p>
<p>Move your .fasta files from the fastq directory into the newly created fasta directory. </p>

[GUIDELINES]
<p><a href="http://bowtie-bio.sourceforge.net/bowtie2/manual.shtml" target="_blank">Bowtie2 Documentation</a></p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Protocol for DNA extraction from clay-rich subsoils
AUTHORS: Lukas Beule

[STEPS]
?. [Sample preparation]
Weigh in 200 ± 5 mg freeze-dried soil into a 2-mL tube.
?. [Subsoil DNA extraction]
1.     Homogenize the sample by bead beating using 3 tungsten carbide beads (ø 3 mm) for 1 min at 25 Hz using a         swing mill (e.g. MM 400, Retsch, Germany).2.     Add 250 µL PB buffer1 with 0.5% SDS (w/v) and vortex for 10 sec at max speed. 3.     Incubate the suspension at 65°C for 10 min and shake the samples every 60 sec for 5 sec.4.     Centrifuge for 1 min at 10,000 rpm and transfer 90 µL of the supernatant into a new 2-mL tube (supernatant will              contain bubbles).5.     Add 810 µL ddH2O (1:10 dilution) and subsequently 900 µL phenol2 and shake the samples briefly.6.     Centrifuge for 10 min at 10,000 rpm and transfer 800 µL of the upper phase into a new 2-mL tube.7.     Add 800 µL chloroform:isoamylalcohol (24:1 (v/v)), incubate 10 min on ice and centrifuge for 10 min at 10,000         rpm.8.     Transfer 700 µL of the upper phase into a new 1.5 mL-tube.9.     Add 700 µL chloroform:isoamylalcohol (24:1), incubate 10 min on ice and centrifuge for 10 min at 10,000 rpm.10.  Transfer 600 µL of the upper phase into a new 1.5 mL-tube containing 200 µL 30% PEG 6000 and 100 µL 5M         NaCl.11.  Shake the mixture and incubate at room temperature for 20 min.12.  Centrifuge for 15 min at max. speed.13.  Wash the pellet with 500 µL 80% EtOH (v/v) twice and centrifuge for 5 min at max speed in-between each         washing before discarding the supernatant. Supernatant should be removed by pipetting.14.  Dry the pellet using vacuum centrifugation at 30°C for 20 minutes or until it is dry under a sterile bench.15.  Add 50 µL TE buffer, vortex or flick the samples to release the pellet from the tube wall and incubate at 42°C for         2h (e.g. in a water bath). Notes addressing the solutions: 1 PB buffer: 1 M Na2HPO4 and 1 M NaH2PO4, blended to achieve pH 7.2.2 Phenol: redistilled, in TE buffer equilibrated, pH 7.5 to 8.0
?. [Protocol source]
This protocol is the Supplementary File S1 of Guerra et al., Microorganisms 2020,8(4), 532; https://doi.org/10.3390/microorganisms8040532please cite as Guerra et al. (2020) Microorganisms,8(4), 532.
TITLE: Hi-Fi Gibson Assembly
AUTHORS: NUS iGEM

[STEPS]
?. Calculate for volumes of respective fragments to assemble based on their length and concentration
?. Add calculated volume of each fragment (maximum volume:  )
5 µl
?. Add in   of 2x Hi-Fi DNA Assembly Master Mix into the PCR tube
5 µl
?. Vortex to mix
?. Spin down PCR tube
?. Incubate samples at  for
50 °C
TITLE: Anti-BrdU Staining Protocols Using DNAse with Surface and Fluorescent Proteins
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"></div>

[STEPS]
?. Pulse actively dividing cells with BrdU (in vitro, cell culture media can be pulsed by adding 10-40µM of BrdU for 1-2 hours).
?. Harvest cells and centrifuge for 5 minutes at 1200-1500 rpm (200-300xg).
?. Wash cells in Cell Staining Buffer (Cat. No. 420201) and centrifuge for 5 minutes at 1200-1500 rpm (200-300xg). Discard supernatant.
?. Aliquot 5 x 105-1 x 106 cells per 12 x 75mm tube
?. Optional: Stain cells for surface antigens if required, utilizing the Cell Surface Immunofluorescence Staining Protocol.
?. Wash cells by adding 1ml of Cell Staining Buffer to each tube and centrifuging for 5 minutes at 1200-1500 rpm (200-300xg). Discard supernatant.
?. Fix cells by adding 100µl of 4% paraformaldehyde at room temperature for 20-30 minutes.
?. Wash cells by repeating step 6 twiceOptional: Cells can be stored in FACS buffer at 4°C for up to 72 hrs
?. Permeabilize cells by adding 500µl of 0.5% Triton-X 100 in PBS for 15 minutes at room temperature.
?. Wash cells by repeating step 6 twice.
?. Treat cells with 20µg of DNAse (Cat. No. D4513, Sigma-Aldrich) diluted in DPBS with calcium and magnesium to each tube and incubate at 37°C for 1 hour.
?. Wash cells by repeating step 6 twice.
?. Add 50µl of Cell Staining Buffer to each tube then add the recommended concentration of anti-BrdU antibody to each tube. Incubate for 20 minutes at room temperature in the dark.
?. Repeat step 6.
?. Stain DNA by adding 1µg of either 7-AAD (Cat. No. 420403) or DAPI (Cat. No. 422801). Wait for 5 minutes prior to acquiring samples on flow cytometer. Note: Adding a 7-AAD or DAPI stain allows you to analyze total DNA content and provides the characteristic horseshoe flow cytometric staining pattern when compared against BrdU. This helps identify the different phases of the cell cycle.