eamag/protocols-with-steps huggingface dataset:
TITLE: Untargeted Top-down Proteomics by LC-MS/MS on Lumos
AUTHORS: Bryon  Drown, Jeannie  Camarillo, Neil  Kelleher

[DESCRIPTION]
Describes the LC-MS/MS data acquisition procedure for top-down proteomics samples using the Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer

[STEPS]
1. Samples were analyzed on a Thermo Scientific Orbitrap Fusion Lumos Tribrid mass spectrometer in line with a Dionex Ultimate 3000 RSLCnano system
2. Samples (6 µL ) were injected via the autosampler and loaded onto a self-packed trap column (150 μm i.d. x 2 cm length packed with PLRP-S 5-μm particles 1,000-Å pore size) for 10 min with 100% loading buffer (94.8% water:5% acetonitrile:0.2% formic acid) 

3. Following a valve switch and initiation of the nanopump at 300 nL/min (buffer A: 94.8 % water, 5 % acetonitrile, 0.2 % formic acid; buffer B: 4.8 % water, 95 % acetonitrile, 0.2 % formic acid), proteins were separated on a self-packed analytical column (75 μm i.d. x 25 cm length packed with PLRP-S 5-μm particles 1,000-Å pore size) according to the following gradient for fractions 1-4:
 
 %BValve position  0  51_1010  51_2121562556795729575  590  5
 
For fraction 5 and later, nanopump used the following gradient:

 

 
4. Eluted proteins were ionized in positive ion mode nanoelectrospray ionization (nESI) using a pulled tip nanospray emitter (15-μm i.d. ×125 mm) packed with 1mm of PLRP-S 5-μm particles 1,000-Å pore size with a custom nano-source (https://proteomicsresource.washington.edu/docs/protocols05/UWPR_NSI_Source.pdf).


 

5. Precursor (intact protein) spectra were acquired at 120k FTRP

 

6. The mass spectrometer was operated using a Top2 data-dependent acquisition mode. Precursor ions were filtered by intensity, charge state, and dynamic exclusion.

 

7. Ions for fragmentations were isolated and fragmented via higher energy dissociation (HCD)
TITLE: Annealing Oligonucleotides
AUTHORS: Brian P P Teague

[DESCRIPTION]
While most biologically-synthesized DNA molecules are double-stranded, chemically-synthesized DNA molecules are single-stranded. In order to use them in a biological system, we need to make them double-stranded by "sticking" them to their complementary molecules:

  

We "stick" them together by mixing them in equimolar amounts, then heating the mixture and cooling them slowly. As the image shows, the duplexed oligos are not a perfect match. Instead, they have four-base "overhangs" on each end -- this will be important in the next protocol.


[BEFORE_START]
Make sure that the thermocycler is programmed with the the annealing program.

[GUIDELINES]
Make sure you take care in your pipetting and mixing. The volumes are small!

[STEPS]
SECTION: Resuspending oligos
1. If the oligonucleotides have already been resuspended, skip to step 5.

Look carefully at the label of the oligo that you're resuspending. Find the line that looks like "14.5 OD = 26.1 nmol = 0.46 mg". (This is an example; the numbers on your tube will be different.) These are three different measurements of exactly how much DNA is in this tube.
SECTION: Resuspending oligos
2. Take the number of nmol and move the decimal point one place to the right. Add this number of microliters of TE to the blue-capped tube with the dried oligo.


 


SECTION: Resuspending oligos
3. Vortex for 15 seconds to fully resuspend the oligo.
SECTION: Resuspending oligos
4. The resulting oligos are at a concentration of 100 micromolar (µM).

SECTION: Diluting oligos
5. Using the TE and the microcentrifuge tubes, dilute the oligonucleotides to 10 micromolar (µM). Make 100 µl of each. Record your dilution calculations in the table below. Ask an instructor or TA to double-check your math before proceeding.


 

 

SECTION: Annealing the oligos
6. In a thin-walled PCR tube, mix:
5 µL of oligo #1
5 µL of oligo #2

Flick the tube several times to mix well, then use the microfuge to spin down the sample.


SECTION: Annealing the oligos
7. Label the tube and record how you labeled it in your lab notebook.
SECTION: Annealing the oligos
8. Find an instructor or TA to help you load the tube into the thermocycler and run the annealing program.
TITLE: Purification of Human K560-GFP motor (without B+R)
AUTHORS: Andrea M. Dickey, Katherine Surridge

[DESCRIPTION]
K560-GFP purification protocol from the Reck-Peterson Lab based on protocol from Nicholas et al. 2014. Edited for protocols.io by Andrea Dickey and Mariusz Matyszewski in 2022 and modified in 2024 for - bind and release by Kavi Rangan and Katy Surridge.

[GUIDELINES]
All protein purification steps should be performed at 4 °C unless noted otherwise. From Ni-NTA step onwards, purification steps should be completed in the dark. 


[STEPS]
SECTION: Expression
1. pET17b-Kif5b(1-560)-GFP-His should be transformed into BL21(DE3)RIPL cells.
SECTION: Expression
2. Make enough LB for at least 2 L of culture.

SECTION: Expression
3. Grow an overnight starter culture.
SECTION: Expression
4. Transfer starter culture into LB. Make sure to add proper antibiotics
Allow it to grow at   until OD600 reaches 0.6-0.8.

SECTION: Expression
5. Chill cells and incubator to 18 °C 

SECTION: Expression
6. Add 0.75 millimolar (mM) and allow it to grow at   

SECTION: Expression
7. Harvest pellets by centrifugation, wash in ice cold PBS and freeze in liquid nitrogen until use. 
SECTION: Purification
8. Resuspend 2 L worth of pellets in 40 mL supplemented with 1 cOmplete EDTA-free protease inhibitor cocktail tablet (Roche) per 40 mL . Also add 1 mL of50 mg/mL . Vortex vigorously to resuspend.
Incubate on ice for 30 min .

SECTION: Purification
9. Lyse the resuspension by sonication on ice with pulse cycles5 s  on and 20 s rest.

SECTION: Purification
10. Clarify by centrifugation 92600 x g, 30 min, 4 °C in Type 70 Ti rotor (Beckman).

SECTION: Purification
12. Incubate the supernatant with Ni-NTA agarose beads washed with the Wash buffer. Nutate for 60 min at 4 °C in the dark (from this step onwards, keep in dark as much as possible).

SECTION: Purification
13. Apply to gravity column and wash 4 times with 10 mLeach time. 

SECTION: Purification
14. Resuspend the beads in 1 mL elution buffer, incubate for 2 min and elute in 0.5 mL increments. Repeat elution step up to 5 times (following green GFP signal)

SECTION: Purification
15. Combine peak fractions and buffer exchange on PD-10 desalting column equilibrated with Storage buffer (BRB80).
SECTION: Purification
16. Peak fractions of the motor solution were flash-frozen at -80 °C  until further use. Bind and release protocol could be used at this stage, see:  dx.doi.org/10.17504/protocols.io.bp2l61xrdvqe/v1

SECTION: Purification
11. During centrifugation, wash each 4 mL  Ni-NTA agarose slurry with 20 mL Wash buffer.
TITLE: Intraganglionic injection of AAV into thoracic dorsal root ganglia in mice
AUTHORS: Seol-Hee Kim, patil, Thomas Taylor-Clark

[DESCRIPTION]
We have developed a novel method of injecting AAV vectors directly into the sensory ganglia, vagal and dorsal root ganglia, of mice. This technique provides for the first time a tool for expression of specific markers in sensory neurons that aid in the tracing of peripheral sensory innervation of organs like the airways and the esophagus.

[STEPS]
1. Mice were anesthetized with a mixture of ketamine (50 mg/kg) and dexmedetomide (0.5 mg/kg) via intraperitoneal injection.
2. Approximately 4 cm of incision was made over a shaved portion of the mouse back region, just around the neck area. 
3. The skin flaps were opened, and the neck muscles were identified. The C7 and T1 vertebra were used as visual guides. 
4. Muscle fibers were separated to get to the T1 and T2 intervertebral space. The nerve bundle that connects to the ganglia sits outside the vertebrae was identified. The corresponding DRGs are tucked inside the vertebrae. 
5. We followed the spinal nerves and identified the beginning of the ganglia.
6. A pulled glass micropipette (~10-15 μm tip diameter) was prefilled with 0.75 mL of AAV9 then injected unilaterally into the T1-T3 DRGs. 
7. After AAV injection, the mice received atipamezole (5 mg/kg) via subcutaneous injection for rapid recovery. The mice were injected with meloxicam (500 mg/kg via subcutaneous injection) as a post-analgesic on the day and 24 h later.
8. 4 weeks later, mice were euthanized by CO2 asphyxiation for tissue collection.
TITLE: How to effectively control vertical collusion in bidding for government investment projects-Based on fsQCA method
AUTHORS: machongsen, Yun Chen, Liang Ou, Wenxi Zhu

[DESCRIPTION]
The impact of collusion during the bidding processes of Chinese government investment projects is a major concern in academic and policy circles, as collusion breeds corruption and destroys the credibility of governments. Furthermore, it negatively impacts successful project completion, leading to cost overruns and the illegitimate enrichment of colluding agents, regardless of the intended social benefits. Using data from 166 selected regional policy implementations as the research sample, this paper uses the fuzzy set qualitative comparative analysis method to conduct a group analysis of typical cases. The purpose of this study is to identify and better understand the cooperative regional policy implementation environments in China and to identify effective methods to improve the governance quality of collusion controls in construction investment project bidding processes. Five key control paths are identified, covering 94% of the cases. It is also found that in lower social collusion situations, reasonable market competition regulations can directly reduce collusive behavior. The research results will help the government to formulate more adaptive control policies and promote high-quality development of government investment projects.

[STEPS]
TITLE: Hepatitis E and Enterovirus Multiplex Nested RT-PCR
AUTHORS: Shannon Fitz, Alex Shaw, Dilip Abraham, michael Owusu

[DESCRIPTION]
For multiplex, nested amplification of Hepatitis E (HEV) and Enterovirus (EV) samples.

Primer sets and target amplicon size

Outer HEV: HEV_38_f and HEV_39_r (538 bps)
HEV_38_f: GARGCYATGGTBGAGAARG
HEV_39_r: GCCATRTTCCARACRGTRTTCC

Inner HEV: HEV_37_f and HEV_27_r (306 bps)
HEV_37_f: GGTTYCGYGCYATTGARAARG
HEV_27_r: TCRCCRGARTGYTTCTTCC

Outer EV: For1_5NCR and Rev7_5NCR (535 bps)
For1_5NCR: CGGTACCYTTGTRCGCCTG
Rev7_5NCR: ATTGTCACCATAAGCAGCC

Inner EV: For2_5NCR and Rev6_5NCR (388 bps)
For2_5NCR: CAAGCACTTCTGTTWCCC
Rev6_5NCR: CCAAAGTAGTCGGTTCCGC

[STEPS]
SECTION: Hepatitis E and Enterovirus Multiplex Nested PCR
1. First round PCR for combined enterovirus and hepatitis E samples
SECTION: Hepatitis E and Enterovirus Multiplex Nested PCR
1.1. 
  

Prepare master mix on ice per number of samples/controls + 10%. Flick master mix gently to mix, and spin down. Aliquot 20 ul into each well, then add 5 ul of template.

 
 
 

SECTION: Hepatitis E and Enterovirus Multiplex Nested PCR
1.2.  Cycling conditions for first round PCR

Flick samples gently to mix, spin down tubes. Amplify samples using the following conditions: 
 
 
SECTION: Hepatitis E and Enterovirus Multiplex Nested PCR
1.3. Second round PCR

  

Prepare on ice per number of samples/controls + 10%. Vortex briefly, and spin down. Aliquot 24 ul of master mix into each well, then add 1 ul of template.    
 
 
SECTION: Hepatitis E and Enterovirus Multiplex Nested PCR
1.4.  Cycling conditions for second round PCR

 Vortex samples briefly, spin down, and amplify using the following conditions:
 
 
SECTION: Hepatitis E and Enterovirus Multiplex Nested PCR
1.5. Check all samples and controls on an agarose gel or a tapestation.
TITLE: Rodent intracerebroventricular AAV injections
AUTHORS: Miguel Chuapoco

[DESCRIPTION]
This abstract outlines the procedure for AAV injections into the lateral ventricle of C57BL/6J mice (intracerebroventricular injections)

[STEPS]
SECTION: Rodent surgery
1. Raise and house mice in compliance with your local Institutional Animal Care and Use Committee (IACUC) and ensure that all protocols and procedures have been approved by the appropriate ethical and regulatory committees. 

Obtain mice from The Jackson Laboratory (male, 6-8 weeks). 

    
SECTION: Rodent surgery
2. Anesthetize mice using isoflurane (5% for induction, 1-3% for maintenance) with 95% O2/5% CO2 (1 L/min).
SECTION: Rodent surgery
3. Head fix mice in a stereotaxic frame, shave head using clippers, and sterilize the area with chlorohexidine. 
SECTION: Rodent surgery
4. Administer 0.05 mL of 2.5 mg/mL bupivacaine subcutaneously above the skull. 
SECTION: Rodent surgery
5. Using a surgical blade, make an midline incision to reveal the skull. Clean the skull of blood and connective tissue and level the head in the stereotaxic frame. 
SECTION: Rodent surgery
7. Drill 2 bilateral burr holes above the lateral ventricles using the following coordinates: 
Anterior-posterior: 0.6 mm posterior to bregma
Medial-lateral: 1.15 mm from the midline
SECTION: Rodent surgery
8. Using a 10 μL NanoFil syringe with a 33 guage beveled needle, aspirate viral vectors (5 x 1010 or 1.5 x 1011 vector genomes in 3-5 μL) using a microsyringe pump.

 

 

 
 
SECTION: Rodent surgery
9. Slowly lower the needle through the burr hole into the lateral ventricle (1.6 mm from the pial surface) and allow the needle to sit in place for approximately 5 minute. Inject viral vector at a rate of 300 nL/min. 
SECTION: Rodent surgery
10. Allow needle to stay in place for 5 minutes after injection has been complete. Slowly remove needle, and suture.
SECTION: Rodent surgery
11. For 5-days post-surgery, mice were treated with 30 mg/kg of ibuprofen and 60 mg/kg of trimethoprim/sulfamethoxazole via water. 
SECTION: Rodent surgery
6. Intraoperatively administer 1 mg/kg of buprenorphine SR and 5 mg/kg of ketoprofen via subcutaneous injection.
TITLE: Multitrim
AUTHORS: Kenji Gerhardt, Kostas Konstantinidis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Multitrim is an Illumina short read trimming pipeline which facilitates effectively and easily trimming either single end or paired end FASTQ data. Multitrim achieves high quality trimming results by automating the detection of Illumina adapter sequences and sequentially applying two independent read trimming tools with complimentary properties. The script additionally produces quality assessment reports for both the input reads and the post-trim reads, creates consistent naming conventions for results, and compresses the trimmed reads to save on space.</div></div>

[STEPS]
?. All contents of this protocol are best viewed in either the attached manual  , or by going to the Multitrim user manual google doc. You can also follow the instructions on this page to achieve the same results..
?. Install the correct version of Anaconda or Miniconda for Python 3.6+ for your system, if it is not already present.Note: Miniconda will take up less space on your computer, and is the recommended method of preparing your computer to run Multitrim, but Anaconda will
?. Copy and paste the following lines of code in your system's terminal to download and install Multitrim and its dependencies:curl https://raw.githubusercontent.com/KGerhardt/multitrim/master/multitrim.yml > multitrim.ymlconda env create -f multitrim.yml
?. Activate the Multitrim environment by entering the following line of code:conda activate multitrim
?. Test if the installation worked by simply typing "multitrim" in your terminal. Usage instructions will appear if multitrim installed successfully.
?. Example usageSingle end:multitrim -u unpaired_reads.fastq.qz -o output_directory --maxPaired end:multitrim -1 forward_reads.fastq.gz -2 reverse_reads.fastq.gz --o output_directory --max
?. Open
TITLE: Preparation and Administration of Systemic 4-AP
AUTHORS: Leonardo A Parra-Rivas

[DESCRIPTION]
Preparation and Administration of Systemic 4-AP

[STEPS]
1. 



4-Aminopyridine was obtained from Sigma-Aldrich (Cat #A78403), and a stock solution was
prepared in saline. Mice (8 weeks old) were given intraperitoneal injections of
soluble 4-AP at a low dose of 2 mg/Kg or equivalent saline. 




2. Animals were monitored every 20 min and presented no sign of convulsive seizure behavior.
After 3 hrs, animals were anesthetized with isoflurane (VetOne Cat#502017),
followed by cervical dislocation. Brains were rapidly removed and drop-fixed in
4% PFA solution for immunohistochemistry or placed on ice for biochemical
assays.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
A simple procedure to conjugate plasmids between bacteria. Conjugation is performed on sterile filters to reduce the number of plates required (5 can easily be fit on one 10 cm plate) and to make suspending the bacteria in liquid simple following conjugation.

[BEFORE_START]
Don't fuck anything up.

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Expression of molecular markers in mouse and human stellate ganglia
AUTHORS: Daniele  Neri, Seoeun  Lee, Alexandre     J Lafond, Lori  Zeltser

[DESCRIPTION]
Protocol for immunohistochemistry

[BEFORE_START]
The human stellate ganglia (SG) were provided by Dr. Olujimi Ajijola at UCLA. The tissue was collected post-mortem from a 44-year-old female donor and dissected immediately after the donor's death.
The dissection procedure of murine SG is described in the "Tissue Harvesting" section of our protocol, "Injection of cholera toxin subunit B in the interscapular brown adipose fat and Stellate Ganglion dissection."
 


[STEPS]
SECTION: Postfixation of the tissue
1. Both mouse and human stellate ganglia (SG) were fixed in 4% PFA in 0.1M PB overnight at 4°C, washed with Phosphate Buffer Saline (PBS) 4°C for at least 1 h
SECTION: Encasing and slicing
2. Ganglia were encased in Optimal Cutting Temperature compound (Tissue-Tek) cut into 10 μm cryo-sections on slides, and stored at -80°C

SECTION: Immunohistochemistry
3. Thaw the sections at room temperature for at least 1 hour
SECTION: Immunohistochemistry
4. Wash in PBS for 30 min twice at room temperature
SECTION: Immunohistochemistry
5. Incubate in blocking buffer (5% donkey serum in PBS with 0.1% Triton X-100) for 1 hour at room temperature
SECTION: Immunohistochemistry
6. Incubate the sections with primary antibodies diluted in blocking buffer overnight at 4C
SECTION: Immunohistochemistry
7. Wash in PBS for 30 min three times at room temperature
SECTION: Immunohistochemistry
8. Incubate the sections with secondary antibodies and DAPI for 1 hour at room temperature
SECTION: Immunohistochemistry
9. Wash in PBS for 30 min three times at room temperature
SECTION: Immunohistochemistry
10. Mount your slides using relevant mounting media and cover slip
SECTION: Imaging
11. Image the sections using a confocal microscope
SECTION: Analysis
12. Analyze the picture using ImageJ/FIJI
TITLE: Isolation of West Nile virus on Vero cell lines
AUTHORS: Anna Nagy

[DESCRIPTION]
West Nile virus (WNV), a  member of the family Flaviviridae, genus Flavivirus is a mosquito-borne emerging pathogen, which is endemic in most part of Europe, especially in the central and southern regions of the continent. The vast majority of human infections remains asymptomatic, though approximately 20% of infected individuals develops milder, influenza-like disease and less than 1% has severe neurological manifestation. Besides the application of  serological and molecular diagnostic techniques, virus isolation and molecular typing of virus isolates are the most important reference laboratory activities. Here we summarize a brief description of our protocol for WNV isolation on cell cultures.

[STEPS]
SECTION: WNV isolation from PCR positive clinical samples:
1. Cell lines: Vero or Vero E6, that are suitable for West Nile virus. Cell culture should be grown in T25 or T75 cell culture flasks.
- Cell culture medium: DMEM (Dulbecco's Modified Eagle Medium) high (4,5 g/L) glucose with L-Glutamine 1000L (Lonza; Catalog #: BE15-604K; ).
- 5% Foetal Bovine Serum (FBS) (Merck; Catalog F7524)
- Antibiotic: Cell Culture Guard, in a dilution 1:100 (ITW Reagents; Catalog A8906,0050)
- Incubator: 37°C, 5% CO2 concentration.
Note: Prior to the virus isolation, cells should be seeded. After 24-48 hours later, when the cell monolayers are  at 80-90% confluence, cell cultures can be used for virus inoculation.
SECTION: WNV isolation from PCR positive clinical samples:
2. Samples: PCR positive clinical specimens:  serum, plasma or urine.
Note: Urine samples must be filtered on 0.22 µm pore size membrane filters and treated with 0.01 M TRIS buffer to adjust the  pH to 7.2 to 7.8 prior inoculation.

SECTION: WNV isolation from PCR positive clinical samples:
3. Add 1 - 2 ml sample to a T75 cell culture flask or 0.5 ml sample to a T25 flask. Incubate at 37°C in 5% CO2 concentration for 90 minutes. 
Note: if the sample volume is insuffiecient, complete the volume with cell culture medium.
SECTION: WNV isolation from PCR positive clinical samples:
4. After the incubation, do not remove the virus inoculum. Add cell culture medium (DMEM) with 5% FBS and cell culture guard in a dilution 1:100.
SECTION: WNV isolation from PCR positive clinical samples:
5. Incubate at 37°C in 5% CO2 concentration for up to 7 days. Daily monitoring of the presence of any cytophatic effects is recommended. 
SECTION: WNV isolation from PCR positive clinical samples:
6. At day 7 cells should be harvested. A blind passage is recommended, using both the cell culture supernatant and harvested cells. 
SECTION: WNV isolation from PCR positive clinical samples:
7. Increased viral load should be checked by real-time RT-PCR method. Ct values of the original clinical sample and cell culture supernatant should be compared.
TITLE: Immunolabeling of a Whole Mouse Brain
AUTHORS: Andrew Recknagel, Kevin Cao, Molly Logsdon, Judith Baka, Naveen Ouellette, Jayaram Chandrashekar

[DESCRIPTION]
Our mice have sparse viral labeling of target neuron populations with genetically encoded fluorescent proteins (FPs). Delipidation ensures that whole brain samples become optically clear and more accessible to antibody labeling. However, the signal from endogenous FPs is often dimmed or quenched during organic solvent delipidation steps. Tissue expansion also dilutes the concentration of fluorophores. To amplify signals of FPs for high resolution, multi-scale imaging, we immunolabel delipidated brain samples.

[BEFORE_START]
If performing the Whole Mouse Brain Delipidation, Immunolabeling, and Expansion Microscopy protocol, start with a brain that has been delipidated using the Tetrahydrofuran and Dichloromethane Delipidation of a Whole Mouse Brain and Aqueous (SBiP) Delipidation of a Whole Mouse Brain protocols.


[STEPS]
SECTION: Immunolabeling of a Whole Mouse Brain
3. Transfer the brain to a small 4 mL vial containing diluted primary antibody in ~ 4 mL PTxw and incubate with rocking, a nutator or shaker, at Room temperature for 11 days. 
10 μg/brain for primary antibody
20 μg/brain for primary conjugated antibody

SECTION: Immunolabeling of a Whole Mouse Brain
4. Transfer the brain to a 20 mL glass vial and wash with PTxw, rocking on a nutator or shaker at Room temperature, and replacing solution for each step:

PTxw for 60 min
PTxw for 120 min 
PTxw for 240 min 
PTxw 240 min
PTxw for 1440 min
PTxw for 1440 min
PTxw for 1440 min

SECTION: Immunolabeling of a Whole Mouse Brain
2. Using a 20 mL glass vial, wash the brain with PTxw, rotating at Room temperature  
SECTION: Immunolabeling of a Whole Mouse Brain
5. Transfer the brain to a small 4 mL vial containing diluted secondary antibody in ~4 mL  PTxw and incubate with rocking at Room temperature for 11 days.
 20 μg/brain for secondary antibody
SECTION: Immunolabeling of a Whole Mouse Brain
6. Transfer brain to a 20 mL glass vial and wash with PTxw, rocking at Room temperature, and replacing solution for each step:

PTxw for 60 min
PTxw for 120 min 
PTxw for 240 min 
PTxw 240 min
PTxw for 1440 min
PTxw for 1440 min
PTxw for 1440 min
SECTION: Immunolabeling of a Whole Mouse Brain
7. Wash the brain with 1X PBS, rocking at Room temperature  .
SECTION: Immunolabeling of a Whole Mouse Brain
1. Start with a whole mouse brain perfused with 4% PFA, post-fixed, and stored in PBS in a 20 mL vial.