eamag/protocols-with-steps huggingface dataset:
TITLE: IMMUNOCYTOCHEMISTRY PROTOCOL
AUTHORS: Scott Vermilyea

[DESCRIPTION]
This protocol details about the Immunocytochemistry.


[STEPS]
SECTION: Procedure
1. Select coverslips previously fixed in 4% paraformaldehyde and subsequently washed and stored in PBS solution.

SECTION: Procedure
2. Permeabilize: 0.2% TX-100 in PBS for 15 min at Room temperature .

SECTION: Procedure
10. Mount coverslips to slide with Vectashield antifade mounting medium.

SECTION: Procedure
9. Wash 3 x 5 min in PBS.

SECTION: Procedure
8. Secondary antibody: mix desired antibody in PBS containing 5% sniper and 0.1% TX-100.

Can add DAPI in secondary solution or as a separate step following washing of the secondary.

SECTION: Procedure
7. Wash 3 x 5 min in PBS.

SECTION: Procedure
6. Primary antibody: mix desired antibody in PBS containing 5% sniper and 0.1% TX-100 overnight at 4 °C(gentle rocking).

SECTION: Procedure
5. Wash 3 x 5 min in PBS.

SECTION: Procedure
4. Block: 50% Sniper in PBS for 15 min at Room temperature .

SECTION: Procedure
3. Wash 3 x 5 min in PBS.

SECTION: Procedure
3.1. Wash 5 min in PBS. (1/3)

SECTION: Procedure
3.2. Wash 5 min in PBS. (2/3)

SECTION: Procedure
3.3. Wash 5 min in PBS. (3/3)

SECTION: Procedure
5.1. Wash 5 min in PBS. (1/3)

SECTION: Procedure
5.2. Wash 5 min in PBS. (2/3)

SECTION: Procedure
5.3. Wash 5 min in PBS. (3/3)

SECTION: Procedure
7.1. Wash 5 min in PBS. (1/3)

SECTION: Procedure
7.2. Wash 5 min in PBS. (2/3)

SECTION: Procedure
7.3. Wash 5 min in PBS. (3/3)

SECTION: Procedure
9.1. Wash 5 min in PBS. (1/3)

SECTION: Procedure
9.2. Wash 5 min in PBS. (2/3)

SECTION: Procedure
9.3. Wash 5 min in PBS. (3/3)
TITLE: 3D printed stereotax multimodal imaging compatibility assessment
AUTHORS: Isabela Zimmermann Rollin, Lucy Liang, David J Schaeffer

[DESCRIPTION]
PET/CT and MRI for multimodal image compatibility.

This protocol is supplementary to the manuscript: 
Liang, L., Zimmermann Rollin, I., Alikaya, A., Ho, J.C., Santini, T., Bostan, A.C., Schwerdt, H.N., Stauffer, W.R., Ibrahim, T.S., Pirondini, E., Schaeffer, D.J., 2024. An open-source MRI compatible frame for multimodal presurgical mapping in macaque and capuchin monkeys. BioRxiv https://doi.org/10.1101/2024.02.17.580767

[GUIDELINES]
The FDG doses are for proof of concept, and even smaller doses of a radioligand may suffice for practical use, depending on exposure and technical considerations.

[STEPS]
SECTION: PET/CT and MRI for multimodal image compatibility
1. Preparation of eye bars and ear bars: With a syringe, fill the ear and eye bars with a small dose (0.05 MBq ear bar, 1.54 MBq eye bar) of [18F] fluorodeoxyglucose (FDG) and seal the chamber with a MRI-compatible nylon screw. Note that these doses are for proof of concept, and even smaller doses of a radioligand may suffice for practical use, depending on exposure and technical considerations.

 

SECTION: PET/CT and MRI for multimodal image compatibility
2. PET Imaging: Place the ear and eye bars in a small preclinical PET/CT scanner (here we used the Si78; Bruker BioSpin GmbH, Ettlingen, Germany) and use the Paravision-360 software package (version 3.2; Bruker BioSpin Corp, Billerica, MA, RRID: SCR_001964) for image acquisition.
SECTION: PET/CT and MRI for multimodal image compatibility
3. CT Acquisition: Acquire a CT scan using the same PET/CT scanner equipped with a Low Dose 1 mm aluminum filter. Here we used a “step and shoot” method with a 0.6-degree gantry step for image acquisition.
SECTION: PET/CT and MRI for multimodal image compatibility
4. Image Reconstruction: Reconstruct the CT images using the filtered back projection algorithm.
TITLE: Salicornia Germination on Agar Plate
AUTHORS: Lina Maria Caceres Leal

[DESCRIPTION]
This method describes the steps to germinate seeds of various Salicornia species under sterile conditions for different purposes; germination of wild-collected seeds, tissue culture and where disease/pest-free plants are required for hydronic experiments. 

[GUIDELINES]
It is advisable to perform all the steps under sterile conditions.

[STEPS]
SECTION: Media preparation
1. Salicornia medium germination. 
One liter of medium is enough for approximately 20 Petri dishes (100mm x 100mm x 15mm).

 


SECTION: Media preparation
2. Store the Petri dishes at room temperature for short-term storage or at 4 °C for long-term storage.
SECTION: Sterilization of seeds
3. Place the seeds in a 2 mL Eppendorf tube. Add 1 ml 70% (v/v) Ethanol and mix for 1 minute.
Remove the ethanol by pipetting carefully to avoid discarding the seeds.
SECTION: Sterilization of seeds
4. Prepare a solution containing 20% (v/v) bleach and 0.2% (v/v) TWEEN 20. Add 1 mL of this solution to the seeds and incubate for 10 minutes, mixing occasionally. After incubation, remove the solution and wash the seeds four times with Milli-Q water. Carefully remove the supernatant by pipetting to avoid discarding the seeds.
SECTION: Sterilization of seeds
5. Place the sterilized seeds on agar plates. Use forceps to place them one by one, or use a 1000 µL pipette to distribute them onto the agar. Cover the Petri dishes with aluminum foil during the first 24 hours. 

If germination is low, add one drop (50 µL) of 500 µM Gibberellic Acid directly onto the seeds and keep them in the dark for 24 hours to enhance germination.
500 µM Gibberellic Acid = 17 mg/100 mL water

Keep the plates in a controlled-environment growth chamber for approximately two weeks. Grow the seeds under a 12-hour light / 12-hour dark photoperiod at 28 °C.

 



SECTION: Transplanting of seedlings
6. Once the seedlings reach a height of 1 cm on the agar plates, they are ready to be transferred to soil or hydroponics.
TITLE: Aggregation of human recombinant alpha-synuclein
AUTHORS: Minee-Liane Choi

[DESCRIPTION]
This protocol describes a method of generating aggregation of human recombinant alpha-synuclein. 

[STEPS]
1. Humanrecombinant α-Syn Monomeric WTα-Syn is purified from Escherichia coli as
previously described in 
 
[Hoyer et al., 2002]. 
2. Aggregation reactions are carried out using a solution of α-Syn 70 micromolar (µM) in 25 millimolar (mM) Tris buffervsupplemented with 100 millimolar (mM)  NaCl, pH 7.4 (in the presence of 0.01 % volume % NaN3 to prevent bacterial growth). 

3. The buffer is freshly prepared before each experiment and passed through a   
syringe filter (Anotop, Whatman) to remove insoluble contaminants. 

4. Prior to incubation, the reaction mixture is ultra-centrifuged at 90k r.p.m. for 60 min at
4 °C  to remove potential seeds. 

5. The supernatant is collected and separated in two fractions: one kept at 4 °C  at all
times until use(monomers), and a second incubated in the dark at 37°C 37 °C and 200 rpm, during 420 min - 480 min hours to avoid fibril formation (monomers+oligomers). 

6. α-Syn is always kept in LoBind microcentrifuge tubes (Eppendorf, Hamburg, Germany) to
limit surface adsorption.
TITLE: Longmire lysis buffer
AUTHORS: Abigail Wells, Linda Park

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lysis buffer recipe (Longmire et al 1997): </div><div class = "text-block">To make 1 liter</div></div>

[STEPS]
?. [To make 1 L of Longmire]
975 ml double-distilled water
?. [To make 1 L of Longmire]
100 ml of 1 M Tris-HCL, pH 8.0
?. [To make 1 L of Longmire]
200 ml of 0.5 M EDTA, pH8.0
?. [To make 1 L of Longmire]
2 ml of 5 M NaCl
?. [To make 1 L of Longmire]
25 ml of 20% SDS (w/v)  Filter the buffer with an autofill PES bottle top filtration device (sterile 500ml, 0.22 µm)
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This week we’re going to take a tour of the server, which will be our home base for the next several weeks. There are important things to know about how to navigate the server, how to use it politely, and how to look at files. We’re mostly going to be accessing the server via the Terminal, and you’ll need to be familiar with Unix commands, which is why you did that Unix activity earlier this week.</p>
<p> </p>
<p>After that, we're going to learn how to assemble reads into longer contigs using a program called IDBA-UD. We'll learn how to do it on a toy dataset, and then you'll do it on your project datasets.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Polyol dehydrogenases are enzymes that convert polyalcohols into sugars, using NAD<sup>+</sup> as hydrogen acceptor. Sorbitol, mannitol or other polyalcohols can be used as substrates, and the activity of these enzymes is assessed by monitoring the production of NADH. This protocol was based on previously published methods (Maret and Auld, 1988; Fernández et al., 1993), and was used to evaluate polyol dehydrogenase activities in the digestive gland of gastropods. With the proper adaptations this procedure can be applied to other dehydrogenases and other organisms.</p>

[BEFORE_START]
<p>Prepare the following solutions:</p>
<p> </p>
<p><strong>Homogenization medium for marine gastropods</strong></p>
<p>(adapted from Stewart et al. 1994 and Moyers et al. 1991)</p>
<p> </p>
<p>Sucrose - 500 mM</p>
<p>Potassium chloride - 150 mM</p>
<p>Ethylenediamine tetraacetic acid (EDTA), sodium salt - 1 mM</p>
<p>Tris-HCl 50 mM pH 7.4</p>
<p> </p>
<p>This medium, without phenylmethylsulfonyl fluoride (PMSF), can be stored at -20º C in aliquots. To avoid the inactivation of PMSF in <em>aqueous solution,</em> add 5 µl of 200 mM PMSF (in absolute ethanol) per ml of medium just before use (final PMSF concentration 1 mM).</p>
<p>                </p>
<p>Good results were also obtained with freshwater and terrestrial gastropods using this high osmolarity medium.</p>
<p><strong> </strong></p>
<p><strong>Substrate solutions</strong></p>
<p>A. 50 mM NAD<sup>+</sup>solution in water (can be stored frozen in aliquots)</p>
<p>B. 57 mM polyalcohol (sorbitol, mannitol or other) in 50 mM glycine/NaOH buffer pH 10.0</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Protocol for preparing seagrass-microbiome samples for DNA extraction
AUTHORS: Cassie Ettinger

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details methods for preparing seagrass-asscociated microbiome samples for DNA extraction.</div></div>

[STEPS]
?. [Weighing samples:]
Place weigh boat on balance and tare.
Before proceeding through wash steps (if necessary), samples should be weighed (this is important if you are considering quantitative PCR or want to standardize sample amounts); prepare balance for  samples by cleaning with 70% ethanol solution and obtain sterile weigh boats.
?. [Weighing samples:]
Place sample in weigh boat using sterile forceps or scoopulas.
Flame sterilize between samples; procedure: immerse in 1) 10% bleach (0.5% NaOCl) 2) water 3) ethanol 4) flame) – attempt to get approx. constant mass across different sample types – e.g. for sediment ~ .
?. [Weighing samples:]
Record sample weight.
?. [Weighing samples:]
Place sample in appropriate tube to proceed-sediment directly into DNA extraction tubes, other tissues into tubes to be washed.
Plant tissues (roots, leaves, rhizomes) can now be directly placed in tube for DNA extraction or can be further processed to get epiphyte washes and/or then further surface-sterilized in the following steps.
?. [Epiphyte washes]
Add  of diluted Redford buffer to samples for  and vortex/agitate.
Using a 1:50 dilution (in autoclaved H20) of Redford buffer solution [  Tris-HCL,   NaEDTA, 1.2% Ctab] from Kembel et al 2014; I used 1.2% Triton-X instead of Ctab as in Kadivar & Stapleton 2003
500 µl
?. [Epiphyte washes]
Pellet in centrifuge @  for .
Centrifuge: 4000 34
?. [Epiphyte washes]
Resuspend pellet in MoBio solution C1 (or equivalent) and proceed with DNA extraction.
?. [Endophyte sterilization]
Rinse tissue with autoclaved H20.
Using the surface cleaning method described in Arnold et al 2007 (Mycologica); complete after washing for epiphytes – or – if epiphytes are not wanted, you can proceed here
?. [Endophyte sterilization]
Immerse tissue in 95% EtOH ().
?. [Endophyte sterilization]
Immerse tissue in 0.5% NaOCl (~10% bleach) ().
?. [Endophyte sterilization]
Immerse tissue in 70% EtOH ().
?. [Endophyte sterilization]
Rinse by with autoclaved H20 ().
?. [Endophyte sterilization]
Repeat step 5 until tissue has been rinsed three times in H20.
We can stop after this step if not proceeding directly to DNA extraction and can freeze tissues.
?. [Endophyte sterilization]
Crush tissue (using sterile centrifuge pestle) & proceed with DNA extraction.
For hardy plant tissues, you can sterilize an electric drill and use the centrifuge pestle as a drill bit to crush tissues – just make sure to clean drill in between samples.
TITLE: Keto Max Science Reviews - How Does It Works For Weight Loss?
AUTHORS: ketosciencereviewsloss  

[DESCRIPTION]
Keto Max Science Reviews - How Does It Works For Weight Loss?


[STEPS]
1. Keto Max Science Reviews - How Does It Works For Weight Loss?
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It moves seriously when it's fueled with unalloyed heat. Weight loss supplements can help you lose redundant water weight. While this may make you feel slimmer and further energetic, it'll not help with your water weight. This form doesn't do that. It doesn't contain any fat. To get the smallest Keto Max Science price of the period, snap any image on this runner. 

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TITLE: Protocol
AUTHORS: Tea Borkowska, Nikoloz Chkhartishvili, Ekaterine Karkashadze, Otar Chokoshvili, Pati gabunia, Lali Sharvadze, Tengiz Tsertsvadze

[DESCRIPTION]
Abstract
Background
Life expectancy and quality of life of people living with HIV have been dramatically improved after introducing antiretroviral therapy, and the prevalence of non-communicable diseases has increased. Several studies have found that hyperglycemia with or without type 2 diabetes was associated with poor outcomes in people living with HIV.
The study’s objective was to determine the prevalence of hyperglycemia and assess its impact on mortality.

Materials and Methods
A retrospective cohort study was conducted among people living with HIV diagnosed in 2012-2018 and followed through 2020 at the Infectious Diseases, AIDS and ClinicalImmunology Research Center in Tbilisi, Georgia. Primary outcomes of interest included the prevalence of hyperglycemia and mortality. Causes of death were classified according to the Coding of Death in HIV(CoDe) protocol.

Results
Our study included 2914 people living with HIV. Two hundred and forty-two (8.3%)patients had hyperglycemia, with an increasing prevalence by age. Three hundred one(9.7%) participants died over the median 3.71 (IQR: 2.14-5.37) years of follow-up. Among these, 139 (46.2%) were due to AIDS-related causes, 123 (40.9%) – were due to non-AIDS causes, and in 39 (12.9%) cases, the cause of death could not be determined. Overall, the cohort contributed to 11,148 person-years offollow-up (PYFU), translating into a mortality rate of 2.70 deaths per 100 PYFU. The mortalityrate was significantly higher among individuals with hyperglycemia - 11.17 deaths per 100PYFU vs 2.07 deaths per100 PYFU amongnormoglycemicpatients(p<0.0001).
Conclusions
Hyperglycemia was associated with increased odds of mortality. Screening andmanagement of hyperglycemia should be integrated into routine HIV clinical services as part of acomprehensivecarepackage.



[STEPS]
1. Prepare database just like one in the Materials section.
2. Analyse the data contained in database.
3. Formulate conclusions.
TITLE: Human urine procurement and processing
AUTHORS: hannah.anvari, asma.giornazi, shriya.shah, maryellen.pavone, francesca.e.duncan francesca.duncan

[DESCRIPTION]
Purpose: This protocol is intended for use in the collection and storage of human urine in a research setting. The protocol details the collection, processing, and long-term storage for downstream analysis alongside other tissues or samples of interest (i.e. follicular fluid, ovary).

[GUIDELINES]
Researchers will adhere to all safety and training protocols required by the institution (Northwestern Medicine/Northwestern
University) including but not limited to:
Biosafety Certification
Bloodborne Pathogens Certification
Collaborative Institutional TrainingInitiative (CITI program) Certification

[STEPS]
1. A urine sample is collected in a standard specimen cup during pre-op from a nurse. Once the urine sample has been collected it is placed immediately on ice following collection. A research coordinator will bring the urine sample to the lab on ice (2-4 °C). Urine samples should be transported in a blue, biohazard labeled cooler bag (Fig. 1A, B).
2.   

3. On arrival the urine sample biospecimen cup is photographed and then aliquoted from the specimen cup (Fig. 1C) into 5 mL cryovials (Fig. 1D) and then stored at -80°C until analysis.
TITLE: Pluripotency markers staining
AUTHORS: Hanqin  Li, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the standard procedure for staining pluripotency markers, e.g. OCT4, SSEA4, alkaline phosphatase, and etc. on human pluripotent stem cells (hPSCs). 

Protocol overview
A. Immunofluorescence staining
B. Alkaline phosphatase staining

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

[STEPS]
SECTION: A. Immunofluorescence staining
1. Wash cells once with PBS
SECTION: A. Immunofluorescence staining
2. Fix cells with 4% PFA at 4 Room temperature for 15 min 

 

SECTION: A. Immunofluorescence staining
3. Wash cells twice with PBS, incubate 5 min  in between washes at Room temperature 

SECTION: A. Immunofluorescence staining
5. Incubate in 3% Bovine Serum Albumin (BSA) for 60 min at Room temperature

SECTION: A. Immunofluorescence staining
6. Incubate with primary antibody (1:200) in 3% BSA at 4 °C 60 min


SECTION: A. Immunofluorescence staining
7. Wash three times with PBS, incubate 5 min in between washes at 4 Room temperature 

SECTION: A. Immunofluorescence staining
8. Incubate with secondary antibody (1:1,000) in 3% BSA at 4 Room temperature for 60 min in the dark and in a humidified chamber.

SECTION: A. Immunofluorescence staining
9. Wash once with PBS 
SECTION: A. Immunofluorescence staining
10. Incubate with 0.1 µg/ml DAPI for 5 min at 4 Room temperature

SECTION: A. Immunofluorescence staining
11. Wash twice with PBS, incubate 5 min in between washes at 4 Room temperature

SECTION: A. Immunofluorescence staining
12. Image cells, seal the plate with parafilm, and wrap with foil for longer storage at 4 °C. Florescence is usually stable for several weeks. 


SECTION: A. Immunofluorescence staining
1.1. Phosphate buffer saline, PBS, pH 7.4

 

SECTION: A. Immunofluorescence staining
4. Incubate in PBST for 30 min at Room temperature to permeabilize cell membrane 

SECTION: A. Immunofluorescence staining
4.1. PBST, pH 7.4

 

SECTION: A. Immunofluorescence staining
2.1. 4% PFA, pH 7.4
 

SECTION: A. Immunofluorescence staining
5.1. 3% Bovine Serum Albumin (BSA)
 

SECTION: B. Alkaline phosphatase staining
13. Wash once with PBS
SECTION: B. Alkaline phosphatase staining
14. Fix cells with cold 4% PFA, 10 min at 4 Room temperature.

SECTION: B. Alkaline phosphatase staining
15. Wash twice with PBS
SECTION: B. Alkaline phosphatase staining
16. Incubate cells with 100 mM Tris, pH 9.5, 10 min at 4 Room temperature

SECTION: B. Alkaline phosphatase staining
17. Add NBT/BCIP substrate in 100 mM Tris, and let reaction develop at 4 Room temperature, avoiding light until color becomes clearly apparent in control cells. 


 

SECTION: B. Alkaline phosphatase staining
18. Wash in PBS and keep in PBS (violet product is soluble in organic solvent).
SECTION: B. Alkaline phosphatase staining
16.1. 100 mM Tris, pH 9.5
TITLE: Imaging axonal calcium dynamics in ex vivo mouse brain slices
AUTHORS: Yan-Feng Zhang, Stephanie J Cragg

[DESCRIPTION]
This protocol describes how to image calcium dynamics in striatal dopaminergic axons in ex vivo mouse brain slices. We imaged calcium transients in response to single and trains (4 pulses, 100 Hz) of electrical stimulus pulses using genetically encoded calcium indicator GCaMP6f expressed in DAT-Cre:Ai95D mice. 

[BEFORE_START]
This protocol was performed in heterozygous DAT-Cre:Ai95D (4-7 weeks) mice. These mice were bred from homozygous DAT-Cre mice (B6.SJL-Slc6a3tm1.1(cre)Bkmn/J, JAX stock number 006660) crossed with homozygous Ai95D mice (B6;129S-Gt(ROSA)26Sortm95.1(CAG-GCaMP6f)Hze/J, JAX stock number 028865). 

 

We prepare ex vivo mouse brain slices by performing steps 1 to 11 from Protocol: Fast-scan cyclic voltammetry to assess dopamine release in ex vivo mouse brain slices. 

[STEPS]
SECTION: Image Acquisition
2. Change the exposure time to reach a frame rate of around 16.6 Hz every 2.5 min using Micro-Manager v1.4. 16.6 Hz frame rate every 2.5 min using Micro-Manager 1.4. 
SECTION: Image Analysis
5. The following steps were performed in MATLAB vR2019b and Fiji v1.5.

Extract ﬂuorescence intensity from the region of interest 25 µm * 25 µm which was 50 µm away from the electrical stimulating electrode tip.
SECTION: Image Analysis
6. After background subtraction, bleach-correct the Ca2+ transients by ﬁtting an exponential curve function through both the baseline (2 s prior to stimulation) and the last 1 s in a 7.2 s recording window.
SECTION: Image Analysis
7. Expressed data as ΔF/F where F is the ﬁtted curve.
SECTION: Image Acquisition
1. Using a x40/0.8 NA water-objective (Olympus UK), position the stimulating electrode on the surface of the brain slice and centre it in the field of view.
SECTION: Image Acquisition
3. Apply electrical stimulus pulses singly and in trains (4 pulses, 100 Hz) using custom-written procedures in Igor Pro 6 (WaveMetrics) and an ITC-18 A/D board (Instrutech).
 

SECTION: Image Acquisition
4. Record changes in fluorescence intensity using custom-written procedures in Igor Pro 6 (WaveMetrics) and an ITC-18 A/D board (Instrutech).