eamag/protocols-with-steps huggingface dataset:
TITLE: Time-lapse killing assay (monolayer - IncuCyte)
AUTHORS: Philippa R Kennedy, Peter Hinderlie

[DESCRIPTION]
Fluorescent target cells are plated in a monolayer in a 96 well plate. Effector cells are added to that plate and time-lapse imaging in combination with fluorescent indicators of cell death reveal the dynamics of target cell death. 

[BEFORE_START]
If using adherent target cells, label them and plate them the day before the assay. 

If using non-adherent target cells, the day of the assay, start by pre-coating a plate with poly-L-ornithine. 

[STEPS]
1. Target cells are fluorescently labelled to differentiate them from effector cells.
4. Enriched NK cells are added to the target cell monolayer at a 2:1 effector:target ratio, bringing the final volume to 200 μL.  
2. Labelled target cells are plated in a monolayer in a 96 well flat-bottom plate (Cat. No: 353072, Corning). Adherent targets are allowed to adhere overnight. Non-adherent target cells can be immobilised on plates pre-coated with 0.01% poly-L-ornithine (pre-coated for 2 h at room temperature; Cat. No. A-004-C, Fisher Scientific). Cell numbers are titrated for each target, but are generally set at 2 x 10 4 cells/well in 100 μL.
5. Plates are placed in an incubator equipped with a time-lapse microscope with 4x, 10x and 20x lenses (IncuCyte Zoom or S3, Sartorius Inc.) for 48 hours. Images are recorded at 30 min intervals. 
3. An indicator of cell death is added to the media (1/1000 dilution; IncuCyte Caspase-3/7 Green Apoptosis Assay reagent, Cat. No:4440, Essen BioScience). 
1.1. Option 1: Target cells are labeled using an amine-reactive dye (CellTrace Far Red Proliferation Kit, Cat. No:C34564, Thermo Fisher) according to the manufacturer's instructions.
1.2. Option 2: Target cells are stably transduced with a red fluorescent protein (IncuCyte NucLight Red Lentiviral Reagent - EF1α Puro, Cat. No. 4625, Essen Bioscience) according to the manufacturer's instructions.
1.3. Option 3: Target cells are stably transfected with a green fluorescent protein (GFP). In this situation, a red cell death indicator should be used (IncuCyte Caspase 3/7 Red Apoptosis Assay Reagent, Cat. No. 4704, Essen BioScience). 
6. Image analysis is performed in the Incucyte software. Live targets (Target cell fluorescence+ Caspase3/7-) are detected at each time point. The percentage of live targets at any given time point is normalized for the number of live targets in each well at the starting time point (t = 0 h), and further normalized to the growth of targets growing alone. This data is then plotted in GraphPad Prism.
TITLE: Fungal Plate Photography
AUTHORS: Craig Bateman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes how to photograph fungal plates.</div><div class = "text-block"><span>This protocol is part of the Bark Beetle Mycobiome (BBM) Research Coordination Network. For more information on the BBM international network: Hulcr J, Barnes I, De Beer ZW, Duong TA, Gazis R, Johnson AJ, Jusino MA, Kasson MT, Li Y, Lynch S, Mayers C, Musvuugwa T, Roets F, Seltmann KC, Six D, Vanderpool D, & Villari C. 2020. Bark beetle mycobiome: collaboratively defined research priorities on a widespread insect-fungus symbiosis. Symbiosis 81: 101–113 </span><a href="https://doi.org/10.1007/s13199-020-00686-9" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://doi.org/10.1007/s13199-020-00686-9</span></a><span>.</span></div></div>

[STEPS]
TITLE: RT-PCR for NoV
AUTHORS: Hengyun Guan, Chunrong Wang, Lanzheng Liu, Guoliang Yang

[DESCRIPTION]
<p>This assay amplified the partial VP1 gene (Region C) of the norovirus genome. The yield products could be sequenced.</p>

[STEPS]
?. [Reagents]
SuperScriptTM III One-Step RT-PCR with PlatinumTM TaqBy Life TechnologiesCatalog #:12574026
?. [RNA extraction]
Extract total RNA of specimens with the MagNA Pure LC Total Nucleic Acid Isolation Kit (Roche, Mannheim, Germany) according to the manufacturer's instructions.
?. [Reaction system：]
The primers (G1-SKF/G1-SKR for GI, COG2F/G2SKR for GII) were derived from the studies of Kojima, S. and Shinohara, M. et al.  AB1The reagent composition
Volume（25 ul）
22×Reaction Mix
12.5 ul&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
3Forward Primer (25 uM)&nbsp;
0.5 ul
4Reverse Primer (25 uM)
0.5ul
5Taq mix
1.0 ul
6Rnase Inhibitor (30-40U/ μl)
0.5 ul
7RNA
3 ul
8DEPC H2O&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
&nbsp;to 25ul &nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;
?. [Amplification]
The amplification conditions were as follows: 42℃ for 30 min; 95℃ for 15 min, 40 cycles of 95℃ for 30 s, 50℃ for 30 s, and 72℃ for 30 s and a final extension of 72℃ for 10 min.
?. [Products]
The expected amplicon size of GI was 330 bp and GII was 387 bp. The yield products were sequenced to validate the the genotype of NoV.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.ed) for additional information regarding this protocol.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Molecular dynamics simulation (Protein-Ligand)
AUTHORS: Hossein Tarrahimofrad, Amir Meimandipour, Sareh Arjmand, Mohammadtaghi Beigi Nassiri, Ehsan Jahangirian, Hossein Tavana, Javad Zamani, Somayyeh Rahimnahal, Saeed Aminzadeh

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The prepared protein and protein-ligand complexes (protease-SDS and protease-tween complexes) were used in MD simulation, performed using GROMACS v 4.6.5 with CHARMM36 all-atom force field. The CGenFF server (https://cgenff.umaryland.edu/) provides topologies and parameters of ligands compatible with the CHARMM36 all-atoms force field. Protein and protein-ligand complexes were soaked in a cubic box of water molecules, and the charges on the protein were neutralized by the addition of Na</span><span style = "font-weight:bold;vertical-align:super;">+</span><span> and Cl</span><span style = "font-weight:bold;vertical-align:super;">-</span><span> ions. The energy of the system was minimised using the steepest descent algorithm to eliminate bad contact and clashes. The NVT and NPT ensembles were used during the equilibration to achieve the desired temperature (373.15 K for protease and 300 K for protease-ligand complex) and pressure (1 bar) for 100 ps and restraint forces of 1000 kJ/mol. Finally, 40 ns MD run were performed in triplicate after releasing all restraints. All bonds were constrained by the LINCS algorithm.</span></div></div>

[STEPS]
TITLE: Fluo-4 Calcium Imaging
AUTHORS: Edinson Lucumi Moreno

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the measurement of the firing activity of cultured iPSC derived neurons. This procedure is used to prepare cultured neurons for Calcium imaging.</div></div>

[STEPS]
?. [Procedure for Calcium Fluo-4]
Prepare  Fluo-4 stock solution by adding  of DMSO to vial.
45 µl
?. [Procedure for Calcium Fluo-4]
Prepare  aliquots of  Fluo-4 stock in PCR Eppendorf tubes and store them at .
5 µl
-20 °C
?. [Procedure for Calcium Fluo-4]
Take one  aliquot of  Fluo-4 and transfer to  of neurobasal medium (work at low light).
5 µl
995 µl
?. [Procedure for Calcium Fluo-4]
Transfer medium from selected wells of the 96 well plate with differentiated neurons to an Eppendorf tube.
?. [Procedure for Calcium Fluo-4]
Wash well by adding  of neurobasal medium, be careful not to detach of perturb the neurons.
150 µl
?. [Procedure for Calcium Fluo-4]
Repeat the washing step 1 more time.
?. [Procedure for Calcium Fluo-4]
Add  of Fluo-4 AM [ in neurobasal medium] into wells.
120 µl
?. [Procedure for Calcium Fluo-4]
Incubate for .
?. [Procedure for Calcium Fluo-4]
Remove the neurobasal medium with Fluo-4 AM, by transferring it to a waste container.
?. [Procedure for Calcium Fluo-4]
Wash well by adding  of neurobasal medium, be careful not to detach of perturb the neuron.
150 µl
?. [Procedure for Calcium Fluo-4]
Remove the neurobasal medium to waste.
?. [Procedure for Calcium Fluo-4]
Add the old differentiation medium to the same well you took them from.
?. [Procedure for Calcium Fluo-4]
Incubate for .
?. [Procedure for Calcium Fluo-4]
Acquire time lapse images in Nikon microscope using the Fluo-4 Calcium imaging acquisition protocol.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This method describes frying protocols for tortilla chips. Chips or other foods are often fried in different oils and oil blends to assess&nbsp; the oxidative stability of the oil, thereby determining whether that particular oil or oil blend is suitable for frying. With this method, one can remove aliquots of oil at any point during the process for storage and/or analysis; just be advised that the oil is hot!

[BEFORE_START]
Be sure your analytical balance will tolerate 900 g of material.<br /><br />Clear the area of flammable items.

[GUIDELINES]
Be sure your analytical balance will tolerate 900 g of material. Ours didn't, so we had to weigh the oil in two 450 g batches. <br /><br />Clear the area of flammable items.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Single-Molecule Antibody Slides For Fluorescence Microscopy 
AUTHORS: Rebecca Andrews

[DESCRIPTION]
This protocol describes how to create single-moelcule antibody slides for fluorescence microscopy. 

[STEPS]
SECTION: Coverslip Cleaning
1. Coverslips (24 x 50 mm, #1, VWR,Catalogue Number 48404-453) were argon plasma cleaned (Ar plasma cleaner, PDC-002, Harrick Plasma) for 30 min .

SECTION: Surface preparation
2. A trimmed gasket was placed on top of the slide (CultureWell‱ Reusable Gasket, 6mm diameter, Grace Bio-Labs, SKU: 103280). 
SECTION: Surface preparation
3. Poly-L-Lysine 30 µL  (0.01 % w/v PLL, P4707, Sigma-Aldrich) was placed in the wells for 30 min. 

SECTION: Surface preparation
4. The PLL was removed, the wells washed three times with PBS (pH 7.4, 1x Gibco, Thermo Fisher Scientific, Catalogue number 10010023) 
SECTION: Depositing Antibodies
5. A secondary antibody of choice was added at a final concentration of 0.0002 mg/ml in PBS. 
SECTION: Depositing Antibodies
6. The antibodies were left in the wells for 5-10 seconds for sufficient surface density before the wells were washed three times with PBS. 
SECTION: Depositing Antibodies
7. PBS 30 µL  was left in the wells for imaging
TITLE: RNA extraction, cDNA synthesis and Taqman qPCR
AUTHORS: gurvir.virdi

[DESCRIPTION]
RNA extraction, cDNA synthesis and TaqMan qPCR on samples.

[STEPS]
SECTION: RNA extraction
1. Cell pellets are snap-frozen using dry ice. 
SECTION: RNA extraction
2. RNA is extracted using the Maxwell® RSC simply RNACells kit (Promega), and the Maxwell® RSC 48 instrument, following manufacturer instructions.
SECTION: RNA extraction
3. After RNA extraction, RNA concentration and quality are measured and assessed using a nanodrop.
SECTION: cDNA synthesis
4. Up to 1 µg of RNA per sample is reverse-transcribed into cDNA using the High-Capacity cDNA Reverse Transcription kit (ThermoFisherScientific).
SECTION: quantitative PCR
5. qPCR is performed using TaqManTM Gene Expression Assay (Thermo Fisher Scientific) according to the manufacturer’s instructions. 
SECTION: quantitative PCR
6. For each gene, TaqManTM probes were used along with the TaqManTM master mix, and sample cDNA following the manufacturer's protocol.
SECTION: quantitative PCR
7. Samples, along with a minus reverse transcriptase control (-RT) were ran for each gene on the QuantSudio 6 Flex Real-Time PCR System (Applied Biosystems).
SECTION: quantitative PCR
8. The -RT served as a negative control, and the gene expression levels were normalised to the housekeeping gene GAPDH following the delta-delta Ct method. Gene expression values were expressed as the normalisation to their respective control sample.
TITLE: No Title
AUTHORS: 

[BEFORE_START]
<strong>What you need before you start:</strong><br />1. 20% nutrient Zobell plates <br />2. Top agar – 3.5 ml per plate<br />       a. 100% nutrient Zobell<br />       b. 6g agar/liter<br />3. Your host grown to exponential phase.  You will need 0.4ml for each plate in the assay.<br />4. Phages to be tested.  This could be<br />          a.  a lysate (0.2 µm filtered)<br />          b.  a plaque picked into buffer<br />          c.  an environmental sample<br />          d.  a sample from an experiment (e.g., a one-step growth curve assay)<br />5.  A water bath set to 40<sup>o</sup>C.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Ef_electocomp_cells_OG1RF
AUTHORS: Elizabeth Fozo

[STEPS]
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Inoculate 5-10 mL BHI or THB (I add fusidic acid at 25 ug/mL but not rifampicin) and incubate o/n at 37°C.
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Dilute o/n culture 1:50 or 1:100 with in 100 mL THB (with FA25) and incubate until culture OD at 600 nm reaches 0.5-1.0 (original protocol dilutes 1:10)
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Chill cells on ice for 15-20 min. Use floor centrifuge to pellet cells (in Oakridge tubes or 50 mL Falcon tubes) at 6,000 rpm for 10 minutes (this step doesn’t need to be at 4 oC).
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Resuspend pellet in 2 mL 10% glycerol or sterile H2O and split into two 1.5 mL tubes. Pellet 13,000 rpm for 1 min. in a tabletop centrifuge (this step doesn’t need to be at 4 oC).
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Resuspend each pellet in 500 µL lysozyme solution (LS) containing 25 μg/ml lysozyme* (add just before use-see recipe in Materials). Incubate at 37°C for 20 min (water bath or incubator).
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Pellet as in step 4.
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Wash 3 times with 1 ml ice-cold electroporation buffer (EB – see recipe below). Keep pellets and buffers cold from this point forward!
?. [E. faecalis Electrocompetent Cells (with Lysozyme)]
Resuspend each tube in 300 uL EB/tube (total from both tubes pooled will be approx. 500-600 uL, including volume of cells) and split into 100 uL aliquots. Store at -80°C. Efficiency will decrease after freezing
?. [Electroporation]
Thaw cells on ice. I use ~50 uL per electroporation.
?. [Electroporation]
Chill DNA and tubes for recovery (1 tube per electroporation). Transfer an appropriate volume of STHB to a test tube (do NOT put on ice). Chill electrocuvettes (1 per electroporation and a few extras, we use 0.1 cm cuvettes).
?. [Electroporation]
Open program on Gene Pulser (Dawn has a standard E. faecalis one saved, settings are .6 kV, 200 Ω, 25 uF).
?. [Electroporation]
Transfer 50 uL cells to the first microfuge tube. Add ice-cold DNA (~100 ng, 1-3 uL of most minipreps will be fine) and pipette a couple of times to mix. Immediately transfer to pre-chilled electrocuvette and electroporate. Immediately add 1 mL STHB to the electrocuvette after the pulse is completed. The time constant should be ~5.0 (higher for pure cells, lower with more or low-quality DNA). Continue even if it’s lower or it arcs. Pipette up and down a few times to wash cells out of cuvette. Transfer back to the microfuge tube you mixed your cells and DNA in during step 2. Keep at room temp/recovery temperature from here on out!
?. [Electroporation]
Recover cells with static incubation at 37°C (or whatever temperature you need) for 2-4 hours
?. [Electroporation]
Plate 50 uL undiluted cells. Pellet the remaining volume and resuspend in 100 uL STHB. Plate 50 uL of the resuspended cells. Save the rest on your benchtop overnight. Incubate plates at 37 oC until colonies appear (1-2 days).
TITLE: Dual-task training to improve gait parameters in people with Parkinson’s disease: a systematic review
AUTHORS: Elisabetta Sarasso, Marco Parente, Federica Agosta, Massimo Filippi, Davide Corbetta

[DESCRIPTION]
People with Parkinson’s disease (PD) frequently exhibit changes in spatiotemporal gait parameters that affect walking abilities. Preliminary studies suggested that dual-task training of gait could enhance walking; nonetheless, it remains unclear whether dual-task training can specifically enhance dual-task gait performance compared to single-task training.
We aim to conduct a systematic review to compare the effect of dual-task training relative to single-task training of gait on specific gait parameters during dual-task tests in subjects with PD.

[STEPS]
SECTION: Search Strategy
1. Preparation of the search strategy and first running on April 2023
SECTION: Records management, selection and data collection
2. Selection and collection starting in January 2024
SECTION: Risk of bias assessment
3. Assessment planned after study selection
SECTION: Data synthesis
4. Depending on data availability; if possible a quantitative synthesis will be performed
TITLE:  human alpha-synuclein and aggregated alpha-synuclein immunofluorescence staining
AUTHORS: Pietro La Vitola

[DESCRIPTION]
This protocol is designed for  human alpha-synuclein staining using the MJFR1-Alexa 488 (RRID:AB_2537217) antibody and either anti-SynO2  (RRID:AB_2632701) or MJFR-14-6-4-2  (RRID:AB_2714215) antibodies. Tissue stained with this protocol include 35 μm free-floating mouse brain sections. All tissue was from mice perfused with 4% PFA. 


[STEPS]
SECTION: Day 1
1. Rinse brain slices (35µ) in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) 3X 10 min 

SECTION: Day 1
2. Quench brain slices in a solution containing 3%H2O2 and 10% Methanol in TBS 20 min   
SECTION: Day 1
3. Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6) 3X 10 min 

SECTION: Day 1
4. Incubate in blocking buffer (5% donkey serum, 2% BSA, 0.5% triton X-100 in TBS) 60 min  hour at room temperature
SECTION: Day 1
5. Incubate in mouse on mouse blocking buffer (MKB-2213; Vector Lab) 20 min at room temperature
SECTION: Day 1
7. Incubate with anti-SynO2 (1:1000; RRID:AB_2632701) or anti-MJFR-14-6-4-2 (1:20000;
RRID:AB_2714215) dilution in 50% blocking solution 2880 min  hour  at 4°C
SECTION: Day 1
6. Rinse brain slices  in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6)10 s 
SECTION: Day 1
8. Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100)) 3X 10 min 
SECTION: Day 1
11. Incubate with rabbit anti-hASYN(1:400; RRID:AB_2537217, MJFR1-Alexa 488) 1440 minat 4°C in 50%  blocking buffer  
SECTION: Day 1
10. Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 3X 10 min 
SECTION: Day 1
12. Rinse brain slices in TBS-T (TBS+ 0.25% Triton X-100) 2x 10 min 
SECTION: Day 1
14. Mount free-floating sections on SuperFrost+ slides (if staining free-floating tissue) and let dry at room temperature for 15 minutes.
SECTION: Day 1
15. Coverslip with fluorescent mounting medium and #1.5 coverslips. Outline coverslip with clear nail polish and store at 4oC .
SECTION: Day 1
9. Incubate in fluorescent secondary antibody at 1:200 dilution in 50% blocking buffer 60 min r at room temperature.
SECTION: Day 1
13. Rinse brain slices in TBS (0.05M Trizma base and 0.15M NaCl; pH: 7.6)10 min
TITLE: Open field
AUTHORS: Miquel Vila, Núria Peñuelas, Ariadna Laguna

[DESCRIPTION]
Open field

[STEPS]
1. Place the mice in a square open field arena of 80 x 80 cm of white methacrylate under brightly lit (300 lux).
2. Track the center and the periphery using a tracking software (SMART 3.0 Panlab-Harvard Apparatus). 
3. Clean the arena every time between animals.
TITLE: Human skin single cell dissociation
AUTHORS: James Fletcher, Rachel Botting, Emily Stephenson, Peter Vegh, Muzlifah Haniffa

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Splitting dermis from epidermis, then dissociating single cells from human skin.</div></div>

[STEPS]
?. Cut skin into thin strips, ~1.5cm wide
?. Stretch the tissue using forceps, and using an 80μm guard on a dermatome cut the top layer of skin. Place this in ice cold PBS
?. Cut a mesh of slits into the 200μm skin layer to aid enzymatic access
?. Place the skin strips in a 50ml Falcon tube in RPMI + 2U/ml Dispase II
?. Invert the tubes to ensure the dispase has sufficient access to all surfaces
?. Seal the tubes with parafilm and place in a 37oC water bath for 1 hour. Invert the tubes every 20 minutes to aid digestion
?. Dunk the skin sheets in PBS to dilute the dispase
?. Use fine forceps to peel the epidermis from the dermis, placing each in PBS in separate containers on ice while peeling
?. Place the epidermis in a cell culture dish with 20ml RF10 (RPMI + 10% FCS (v/v) + 100U/ml Penicillin + 100μg/ml Streptomycin + 1% L-Glutamine (v/v). Add 1.6mg/ml type IV Collagenase. Place this in a 37oC 5% CO2 incubator overnight (~12 hours)
?. Repeat step 9 with the dermis
?. Collect the supernatant with a serological pipette and pass it through a 100μm cell strainer, keeping the dermis and epidermis separate
?. Wash the base of the culture dish with RF10 to collect any settled cells and pass this solution through a 100μm cell strainer
?. Centrifuge the collected cells at 500g for 5 minutes (high acceleration/deceleration) and resuspend in PBS to count
TITLE: Buffer preparation for OnePot PURE cell-free system 
AUTHORS: Konstantinos Ragios

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">In this protocol we explain the procedure to create the Buffers used for Protein and Ribosome purification for the production of OnePot PURE cell-free system. </div></div>

[STEPS]
?. Add the materials needed the buffer you want to produce in a beaker. The final concentration of the components for the different buffers is presented in Table 1 and Table 2
?. Table 1:  Buffers for Protein Purification  ABCDE1CompoundBuffer ABuffer BBuffer HTStock buffer B2mMmMmMmM3HEPES505050504Ammonium chloride10005Magnesium chloride101010106Potassium chloride1001001007Imidasol5008Glycerol60%9β-mercaptoethanol7777
ABCDE1CompoundBuffer ABuffer BBuffer HTStock buffer B2mMmMmMmM3HEPES505050504Ammonium chloride10005Magnesium chloride101010106Potassium chloride1001001007Imidasol5008Glycerol60%9β-mercaptoethanol7777
In Stock Buffer B, Glycerol should consists of 60% of the final volume
?. Table 2: Buffers for Ribosome Purification   ABCDEFG1CompoundSuspension bufferSuspension buffer high saltBuffer CBuffer DCusion bufferRibosome buffer2mMmMmMmMmMmM3HEPES1010202020204Magnesium acetate101010101065Potassium chloride5050306Ammonium chloride307Ammonium sulfate300015008Sucrose30%9β-mercaptoethanol777777
ABCDEFG1CompoundSuspension bufferSuspension buffer high saltBuffer CBuffer DCusion bufferRibosome buffer2mMmMmMmMmMmM3HEPES1010202020204Magnesium acetate101010101065Potassium chloride5050306Ammonium chloride307Ammonium sulfate300015008Sucrose30%9β-mercaptoethanol777777
In Cusion buffer, Sucrose should consists of 30% of the final volume
?. Add some distilled water and mix with a magnetic stirrer until all the solid components are dissolved
Be careful so the volume at this point does not surpass the desired one, otherwise the components' concentration will be wrong on the final product.
?. Add as much distilled water is needed to reach the final volume of the buffers
?. Filter the solution.
?. The buffer must be stored at 4oC
?. Because β-mercaptoethanol has a short half-life we need to add it again to the buffer every time before we perform an experiment.
?. The buffers are then stored at 4oC
TITLE: Expression of molecular markers in subpopulations of mouse stellate ganglion neurons
AUTHORS: Daniele  Neri, Lori  Zeltser

[DESCRIPTION]
This protocol is a direct application of the original RNAScope Fluroescent Multiplex Reagent Kit (ACDBio, Document Number 320293-USM, https://acdbio.com/sites/default/files/320293_RNAscope%20Multiplex_UM_04102019%20%281%29.pdf)

[STEPS]