eamag/protocols-with-steps huggingface dataset:
TITLE: Electrolysis agarose gel preparation
AUTHORS: UM ILT team, Mar Roca Cugat

[DESCRIPTION]
This is how students at Maastricht University's Biomedical Sciences bachelor are instructed to prepare agarose gel for electrolysis. 

[STEPS]
1. Prepare the casting tray by putting the sides together.
2. The silicon coated site should point towards the inside.
3. Apply the elastic band around the extruding parts in the middle of the sides to hold everything in place.
4. Flip the assembled tray in the correct position and press it lightly on the table to ensure proper alignment of the both sides.
5. Add the comb and put the tray in the box to catch any potential spills if not correctly assembled.
6. Prepare a 1% agarose gel in 1x TAE buffer. The total volume depends on the size of the casting tray; we will make 100 mL gels. Calculate and weigh the amount of agarose required for your volume. Deposit the agarose in a glass Erlenmeyer flask and add the indicated volume of 1x TAE buffer.
7. Pour the mixture in the casting tray with combs. Remove any air bubbles with a plastic filter tip.


8. Allow the gel to solidify (10 min). 

8.1. Remove the comb.
9. Insert the casting tray into the electrophoresis equipment; the side of the slots in the direction of the cathode (black). 
Do not add the metal casting system into the electrophorese system, just the gel.
10. Prepare and add 1x TAE Buffer to the system until the gel is completely submerged.
TITLE: SEM User Protocol
AUTHORS: Elizabeth Fozo

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">How to use the Auriga Scanning Electron Microscope at JIAM</div></div>

[STEPS]
?. [How to:]
After samples have been prepped, and the silicon chip is placed on carbon tape sticking it to the aluminum stub, grab a sample holder in the desiccator vessel usually on the backbench in the Auriga room.  The sample holder has 3 screw holes on one side and what looks like a bracket on the base.  Use the fallen key to secure your samples onto the holder – do not over tighten.
?. [How to:]
At the machine, press Vent first – may hear some gas sound.
?. [How to:]
Pull open the door gently.
?. [How to:]
Put the sample holder onto the dock and then secure the slide bar to the sample holder.
?. [How to:]
Close the compartment and then press Store then press Exchange on the keyboard.
?. [How to:]
The Transfer button will blink and when done will remain solid green.
?. [How to:]
Push the large rod into the chamber – there will be some resistance when the sample holder is on the stage – push gently and then once in place, unscrew the rod from the sample holder – be sure to look at the monitor to ensure that the sample is correctly positioned and is not moving when you start to unscrew the rod.
?. [How to:]
After the mount is in place and the rod has been removed hit Resume on the keyboard.
?. [How to:]
Start to use the two joysticksThe small joystick will move the stage up and down as adjust your working distance – normally you want around 10.  It will also tilt the stage.  Tilting the stage is not necessary.The large joystick will move the stage in the X and Y plane.  It can also rotate the stage.  This is critical for aligning your sample under the electron beam. Typically after you find your first sample, you can just rotate to find your next.
?. [How to:]
In the lower right of the user interface, make sure that EHT is on.  A green checkmark lets you know you are good.
?. [How to:]
Double click on the kV = 5 should be good. Having a high kV improves the resolution of the sample, but it also increases the charging – makes sure you use a WD of about 10.
?. [How to:]
Brightness should be around 48-49 percent
?. [How to:]
Contrast just mess around with it
?. [How to:]
When done, hit Exchange on the keyboard – wait for the internal door to open – retrieve sample with screw arm.
?. [How to:]
Look for the door to close.
?. [How to:]
Then hit vent.
TITLE: Large Volume Immunostaining for Cleared Samples
AUTHORS: Seth Currlin, Marda Jorgensen, Jerelyn Nick

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This is a guide for immunostaining CLARITY-processed samples. </span><a href="https://dx.doi.org/10.17504/protocols.io.8jihuke" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">dx.doi.org/10.17504/protocols.io.8jihuke</span></a></div><div class = "text-block"><span>These steps are meant to be a guide for immunostaining large samples and should be optimized to suit your particular tissues and reagents. Large tissue volumes and dense tissue types will require longer incubation and wash times. The parameters suggested below are for a piece of human tissue (thymus, spleen, or lymph node) approximately 5 mm</span><span style = "vertical-align:super;">3 </span><span>in size.</span></div><div class = "text-block">A useful link: http://wiki.claritytechniques.org/index.php/Immunostaining </div></div>

[STEPS]
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Wash out residual clearing solutionThree washes in PBST, 24 hours each, 37ºC with gentle agitation. Large wash volumes are best, a 50 ml conical tube with 50 ml fresh PBST is recommended for each wash step.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Nonspecific Epitope BlockingBlocking buffer incubation: three days, 37ºC with gentle agitation. Blocking buffer volume should be at least 5 volumes greater than tissue volume.Note:  Serum type should be used based on the secondary antibody host species.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Primary Antibody Incubation1:100 dilution (~10 ug/mL) in Staining Buffer.Primary antibody incubation: five days, 37ºC with gentle agitation; then two days, 4ºC with gentle agitation. For best results a large volume for primary antibody incubation is recommended however. A 5 ml total volume for antibody staining is recommended for a 5 mm3 piece of tissue.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Wash out Primary AntibodyThree washes, 24 hours each, at 37ºC with gentle agitation.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Secondary Antibody Incubation1:200 dilution (~5 ug/mL) in Staining Buffer. Secondary antibody incubation: five days at 37ºC with gentle agitation; two days at 4ºC with gentle agitation. Note: Protect samples from light.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Wash out Secondary AntibodyThree washes, 24 hours each, at 37ºC with gentle agitation. Note: Protect samples from light.
?. [Immunohistochemistry for large (5 mm3) tissue volumes]
Prepare for 3D Imaging or StoragePrepare for imaging or store samples in washing buffer (PBST) at 4ºC. For 3D imaging guide, see: https://dx.doi.org/10.17504/protocols.io.begajbse
TITLE: Fragment Analyzer Operation for PCR Products
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes fragment analyzer operation for polymerase chain reaction (PCR) products. The Fragment Analyzer by Advanced Analytical is used as a quality control step for genotyping PCR products.  Peak size, shape, and sample concentration are recorded and used to evaluate samples on a pass/fail level.</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: tomato hypocotyl grafting
AUTHORS: hengliu 

[STEPS]
?. Tomato (Solanum lycopersicum) cultivar Zhongshu No.4 seeds were surface sterilized with 75% ethanol for 1 min, and in sterilization solution containing 35% commercial bleach solution (5.25% [w/v] sodium hypochlorite) and 0.1% Tween-20 (Bio-Rad, Shanghai, China) for 6 min.
?. The seeds were placed in 1/2strength MS medium containing 0.8% agarose.
?. The germinated seedlings were grown for 5 to 7 d before grafting in growth chambers and maintained under 16 h of light at 25°C and 8 h of dark at 18°C and 60% relative humidity.
?. Plates were tilted using a glass rod of 0.7–1 cm in diameter.
?. Seedlings were chosen with long straight hypocotyls and the hypocotyl was cut transversely while on the agar.
?. Cut the petiole to the middle part of the hypocotyl flat as a scion, take another seedling that is relatively straight and the hypocotyl diameter is similar to that of the scion, and cut the upper 1/3 of the hypocotyl as the rootstock.
?. Forceps were used to keep the seedling stable while cutting the hypocotyl with the razor blade under a dissecting microscope,It was important to make the cut quickly and to push the blade rather than pull it, so that the cut surface was clean and smooth.
?. The scion was lifted up to bring it to the stock, and the stock was carefully picked up to approach the scion and connect them together. Note that it is important to be aware of raising the graft union up away from the agar surface (Due to the large volume of tomato seedlings, in order to prevent the interface part from being difficult to heal due to contact with the culture medium, a hole can be dug in the culture medium below the interface part with tweezers to suspend the interface part).
?. The scion or stock was carefully and slightly pushed to adjust the relative position of the two parts, and the graft was inspected from all sides of the graft junction to make sure that the two parts connected and supported each other thoroughly.
?. The plate was returned to the growth room to the same vertical position with the thin side downward and the thick side upward.
?. The grafted tomato seedlings were placed in the growth chambers upright for  0, 0.5, 1, 2, 6, 12 h and inspected regularly.
?. The tomato seedlings grafted at different time points were assessed under a microscope, the plants were gently moved with tweezers, and only those plants that were not separated from the junction were used as samples for successful grafting.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a simple PCR-SSP for amplification of the rs6656401, located in intron 4 (CR1 gene).</p>

[BEFORE_START]
<p>1- Wear clean gloves;<br />2- Clean pipettes and stand with hypochlorite and 70% alcohol;<br />3- Defreeze DNA samples and reagents (exception: Taq polymerase);<br />4- Gently mix the DNA samples and pass the reagents briefly on the vortex.<br />5- Centrifuge the DNA samples and reagents with a spin;<br />6- Keep reagents on ice;<br />7- Leave microtubes ready for preparation of the mix, keep the same ones identified and on ice.<br />8- Leave application plates properly identified.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Generation of hiPSC derived cortical astrocytes
AUTHORS: Minee-Liane Choi

[DESCRIPTION]
This is a modified protocol that describes how to generate cortical region-specific astrocytes based on Gupta et al., 2012, Serio et al., 2013 and  Seto-Salvia et al., 2021


 

 

 


[STEPS]
SECTION: hiPSC culture
1. hiPSCs are maintained on Geltrex in mTeSR (Stem Cell) and passaged using 0.5mM EDTA, as followed by Virdi et al., 2022.
 

SECTION: Differentiation of hiPSC into Cortical astrocytes
2. Differentiation of cortical region-specific astrocytes is performed using a modified protocol based on Gupta et al., 2012, Serio et al., 2013 and Seto-Salvia et al., 2020.


 

 

SECTION: Differentiation of hiPSC into Cortical astrocytes
3. Neural precursor cells (NPCs) are obtained from hiPSC using the established protocol of 
 

SECTION: Differentiation of hiPSC into Cortical astrocytes
3.1. First, NPCs are induced by dual SMAD inhibition for 10 days, followed by culturing with the N2B27 media for another 15 days.
 
 
SECTION: Induction of glial precursor cells (GPCs),
4. NPCs are used to derive glial precursor cells (GPCs) by culturing in an N2B27 medium supplemented with 20 ng/ml of human FGF-2. 

SECTION: Induction of glial precursor cells (GPCs),
4.1. Cells are split twice per week (1:2 or 1:3) using Accutase (Cat No. A1110501, Thermo Fisher Scientific) by vigorously breaking pellets to remove neuronal cells. 
SECTION: Final differentiation into astrocytes
5. Upon the appearance of glial morphology (around day 90 from the neural induction), the GPCs are cultured for 7 days with 10 ng/ml Bone Morphogenetic Protein 4 (BMP4) and 20 ng/ml Leukemia inhibitory factor (LIF), which activates the JAK/STAT signalling pathway, refreshing the medium every other day
SECTION: Final differentiation into astrocytes
5.1. On the 8th day, BMP4 and LIF are withdrawn. 
SECTION: Final differentiation into astrocytes
5.2. Cells are further differentiated for the final maturation in the N2B27 media without FGF2. 
SECTION: Final differentiation into astrocytes
5.3. 3-5 times further passes might be required (1:2) until the complete loss of the precursor property.
TITLE: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
AUTHORS: Dan Dou, C. Alexander Boecker, Erika L.F. Holzbaur

[DESCRIPTION]
Here, we describe procedure and equipment used for live-imaging of axonal cargoes. This was performed both using primary mouse cortical neurons and human iPSCderived excitatory glutamatergic neurons. Equipment and software used varied based on laboratory site and scheduled upgrades to microscopy equipment during the course of this study.

[GUIDELINES]
Citations:

Boecker, C.A., Olenick, M.A., Gallagher, E.R., Ward, M.E., and Holzbaur, E.L.F. (2020). ToolBox: Live Imaging of intracellular organelle transport in induced pluripotent stem cell‐derived neurons. Traffic 21, 138–155.
Kaech, S., and Banker, G. (2006). Culturing hippocampal neurons. Nat. Protoc. 1, 2406–2415.

[STEPS]
SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
1. 
 

Image primary mouse cortical neurons on DIV7. Image human iNeurons on DIV21.
SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
2. Replace culture media with low fluorescence imaging media.
SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
2.1. 
For primary mouse neurons, use Hibernate E medium supplemented with

 

SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
2.2. 
For iNeurons, use Hibernate A medium supplemented with

 

SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
3. Image using spinning disk confocal microscope under 60x magnification (oil immersion objective). 
 

SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
4. Identify axons of transfected neurons based on morphological parameters. (Boecker et al., 2020; Kaech and Banker, 2006). For example, axons can most reliably be identified by their length and should span over at least 500 µm. 
SECTION: Live-imaging of axonal cargoes in human iPSC-derived neurons or mouse primary neurons
5. Acquire time lapse recordings at a frame rate of 1 frame per second for 5 min.
TITLE: f/2 medium with soil extract
AUTHORS: Roscoff Culture Collection

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Medium to grow phytoplankton species in particular dinoflagellates which often require soil extract.</div></div>

[STEPS]
?. [Prepare f/2 medium]
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?. [Add soil extract]
{"blocks":[{"key":"e5mbp","text":"How to prepare soil extract","type":"unstyled","depth":0,"inlineStyleRanges":[],"entityRanges":[],"data":[]}],"entityMap":[]}
TITLE: Restriction Digest - OpenPlast
AUTHORS: yasoo , New England Biolabs

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The following is a "typical" restriction endonuclease reaction. Please see the "guidelines" tab below for the NEB tips on optimizing restriction digests.</div></div>

[STEPS]
?. Set up the following reaction (total reaction volume 20µl).  AB1Restriction Enzyme0.5µl2DNA0.5 - 1 µg310X NEBuffer2 µl (1X)4Total Reaction Volume20 µl5Incubation Time1 hour6Incubation TemperatureEnzyme dependent
AB1Restriction Enzyme0.5µl2DNA0.5 - 1 µg310X NEBuffer2 µl (1X)4Total Reaction Volume20 µl5Incubation Time1 hour6Incubation TemperatureEnzyme dependent
Enzyme volume should not exceed 10% of the total reaction volume to prevent star activity due to excess glycerol.
Temperature for Enzymes:BsaI: 37°CBsmBI: 55°CEsp3I: 37°C
The enzyme should be the last component added to reaction
Keep Enzyme on ice when not in freezer.
?. Mix components by pipetting the reaction mixture up and down, or by "flicking" the reaction tube.
?. Quick ("touch") spin-down in a microcentrifuge. Do not vortex the reaction.
?. Incubate for 1 hour at the enzyme-specific appropriate temperature.
Can be decreased to 5-15 minutes by using a Time-Saver™ Qualified enzyme.
See the NEB Activity/Performance Chart for the incubation temperatures.
TITLE: in situ hybridization performed on the peri-rhopalial tissue of a scyphozoan jellyfish
AUTHORS: Christelle Bouchard

[STEPS]
?. 
?. 
?. Fix tissue 2 h, RT, in freshly prepared 4% PF in 0.1 M PB (pH 9.5) + 0.42M NaCl + 2 mM MgSO4
?. Wash 3X 5’ in PBST, pH 7.5
?. Dehydration in PBS/methanol 3:1 → 1:1 → 1:3 10 min each then 100% methanol 5 min to ON in -20C.
?. Rehydration in PBS/methanol 1:3 → 1:1 → 3:1 10 min each then PBST, 10 min.
?. Heat treatment in 1 ml PBST, 5 min. Bain-marie at 95C with stopper. First 2 min, tissue should be resuspended vigorously every 20 sec, every 60 sec during the rest of incubation in order to prevent glueing of specimens. Snap-cool tubes on ice.
?. Wash 2X in PBST
?. Incubation 5 min in TEAA
?. Wash 2X 10 min in PBST
?. Post-fixation in 4% PF in PBST pH 7.5 for 20 min.
?. Wash 3X in PBST, 10 min each
?. Saturation of unspecific binding with 75 ul PBST/HS-DNA (0,5 mg/ml) for 10 min
?. Addition of the same amount of HybMix, incubate 5 min
?. Prehybridization in 150 ul HybMix for 10 min RT
?. Prehybridization in 150 ul HybMix for 2h at 45C
?. Hybridization in 1 ml HybMix + DIG-probes sense or antisense for 1 day and 2 nights at 40C. Before use, denature probe in HybMix at 70C for 10 min and snap-cool on ice.
?. 2 wash in 1 ml of Wash I 60 min at 45C
?. 4 wash in 1 ml of Wash I, 15 min at 45C
?. Wash in PBST 20 min.
?. Saturation in PBST/10% NGS, 2h in fridge with shaking
?. Incubation in anti-DIG-AP-conjugated antibody diluted 1:2000 in PBST containing 1 % NGS/ 4C over the weekend with shaking. (preadsorption was not necessary).
?. Wash 4X in PBST for 20 min.
?. Incubation 3X 10min in AP-Buffer (containing 10mM MgCl2)
?. Substrate reaction with NBT/BCIP solution
?. Stop reaction with PBST/50 mM EDTA. 3X 5min
?. 
?. 
?. Phosphate Buffer pH 9-9.5: made from phosphate dibasic (No pH adjustment required )
?. PBS: 100 mM pH 7.5, 150 mM NaCl
?. PBST: PBS +0.1% Tween
?. 
?. 
?. TEA: 0.1 M triethanolamine. Dissolve powder in water. Adjust pH at 7.8 with 5 N NaOH.TEAA: 5 ml TEA + 12, 5 ul glacial acetic acid. Acetic acid must be added to TEA just before the incubation with tissues.
?. 
?. 
?. 
?. PBST/fish sperm: 0, 5 mg/ml fish sperm DNA in PBST. This DNA was made from herring and cod sperm and has a molecular weight distribution of 150-3000 bases.
?. 
?. 
?. HybMix: 50% formamide 5 x SSC 1mg/ml HS-DNA 0, 1% Tween 20
?. Wash I: 50% formamide 2 x SSC 0, 1 % Tween 20
?. Blocking solution: 10 % normal goat serum in PBST
TITLE: Freezing of hPSC
AUTHORS: Valeria Fernandez Vallone, Lyn Healy, Nathalie Lefort, Katarzyna Ludwik, Tamer Onder, Lisa Pavinato, Fatma Visal FVO OKUR, Harald Stachelscheid

[DESCRIPTION]
This protocol describes cryopreservation of hPSC as clumps or single cells.

[BEFORE_START]
Prepare labelled or barcoded cryo-vials.

[GUIDELINES]
hPSC culture quality is highly relevant for a successful freezing/thawing procedure. 
We recommend freezing cultures at ~70-80% confluency. High quality cultures show spontaneous differentiation below 5-10% of cultured surface. 







[STEPS]
SECTION: Clump freezing
4. Aspirate and discard medium from the vessel.
SECTION: Clump freezing
6. Add required volume of non-enzymatic dissociation reagent to the culture vessel, refer to Table 1 for recommended volumes.
SECTION: Clump freezing
8. Gently aspirate non-enzymatic dissociation reagent gently without disturbing the cells and discard.
SECTION: Clump freezing
10. Gently tap the plate 5 s to dislodge the cells from the plastic surface.
 

SECTION: Clump freezing
12. Immediately place cryo-vials into the freezing container and place at -80 °C   
 

SECTION: Clump freezing
13. Transfer hPSC containing cryo-vials to a liquid N2 tank after 1440 min
SECTION: Clump freezing
9. Add required volume of freezing medium to the culture vessel, refer to Table 1 for recommended volumes.
SECTION: Clump freezing
11. Use a 2 mL pipette to transfer hPSC suspension (ideally 1 mL) into pre-labeled cryo-vials.
 

SECTION: Preparation
3. Choose freezing method:
SECTION: Preparation
1. Visually inspect hPSC culture using a microscope. Culture ready for freezing should be of good quality (see Guidelines section).
SECTION: Clump freezing
5. Rinse once with required volume of non-enzymatic dissociation reagent, refer to Table 1 for recommended volumes.
 

SECTION: Clump freezing
7. Incubate for 3 min at Room temperature. 
 

SECTION: Preparation
2. Refer to Table 1 to prepare adequate amount of dissociation reagent and freezing medium according to the culture vessel format and amount of vessels/wells to be frozen.
 

 

 

SECTION: Single-cell freezing
4. Aspirate and discard medium from the vessels.
SECTION: Single-cell freezing
5. Add required volume of enzymatic dissociation reagent, refer to Table 1 for recommended volumes.
 

SECTION: Single-cell freezing
6. Incubate for 5 min at 37°C.
 

SECTION: Single-cell freezing
7. Collect hPSC suspension in 15 or 50 mL tube containing 5 mL hPSC culture media and rinse wells/vessels with additional culture medium, refer to Table 1 for recommended volumes.
SECTION: Single-cell freezing
8. Centrifuge hPSC suspension at 300 x g  for 5 min
SECTION: Single-cell freezing
9. Aspirate and discard supernatant.
SECTION: Single-cell freezing
10. Re-suspend cells in freezing medium and perform hPSC counting.
 

SECTION: Single-cell freezing
11. Distribute homogeneously among cryo-vials hPSC suspension.
SECTION: Single-cell freezing
12. Immediately place cryo-vials into the freezing container and store at -80 °C 
 

SECTION: Single-cell freezing
13. Transfer hPSC containing cryo-vials to a liquid N2 tank after1440 min
TITLE: Gene set enrichment analysis
AUTHORS: Peter Kilfeather

[DESCRIPTION]
Gene set enrichment analysis from Kilfeather, Khoo et al., 2024

[STEPS]
SECTION: Protocol
1. No statistical methods were used to predetermine sample sizes, but our sample sizes for TRAP analyses surpass those reported in previous publications23,28,87 and our sample sizes for Stereo-seq samples are comparable with or exceed those of similar spatial transcriptomic datasets11. All statistical analyses were performed with R (v4.2.1) and Python (v3.9). All P values were modified to an FDR of 1, 5 or 10 % as described in the text with the Benjamini & Hochberg method.