eamag/protocols-with-steps huggingface dataset:
TITLE: Collecting needle and branch samples for terpenoid and expression analysis
AUTHORS: Vidya S Vuruputoor, karlfetter, victoriaburton, Jill Wegrzyn

[DESCRIPTION]
This protocol is used by the PCG group for sampling needle and branch tissue from hemlock conifers for terpenoid analysis, as well as RNA expression analysis.

[STEPS]
1. Open Treesnap (https://treesnap.org/), record the accession number on the tree and fill in notes for hemlock woolly adelgid infestation (HWA) levels (scale provided by the app).

  

1.1. Use the comment field to enter: EHS present OR EHS absent to record the hemlock scale.

  

1.2. Take at least 3 photos with TreeSnap. This includes at least one of the trees itself, one photo of the tree tag if that is present, and one photo of the collection vial with the label as well.
1.3. Geolocation will be recorded automatically but TreeSnap can also connect with a more precise external GPS device.
1.4. Note DBH in appropriate column
1.5. Note any other observation you may have- including plant condition (Excellent/Good/Poor), and placement of individual (lower canopy, full sun, etc.)
2. If gathering samples for pooled data, choose four random areas around the tree to collect needle and branch samples
 

3. Look for the second branch from the shoot tip (stick to the right first) and snip off 5 cm to place in 10 ml cryovial
4. Repeat for the other 3 directions (check appropriate column in the sheet provided).  Place about 3-4 stems in each 10 ml cryovial.
5. Close cryovial, and using the appropriate gloves to handle liquid nitrogen (LN), place cryovial in LN dewar.


 

5.1. Cap vial tightly to prevent opening in the LN.
5.2. Depending on dewer configuration, you can tie tubes with string to a ‘halo’ to help keep vials from falling to the bottom. Fully submerged tubes can open.  

 


6. Clean trimmers between trees with ethanol (spray bottle).

 

7. Use clipboard to manually record notes on a pre-printed spreadsheet with sample names.
8. After returning from the field, ensure that notes are consistent with TreeSnap entries.  These entries can be amended after collection as needed.
TITLE: Assessment of Knowledge, Attitude and Practice toward Hepatitis B virus infection among Healthcare Providers working at sixty Public and Private Hospitals in Khartoum State Sudan 2023 
AUTHORS: essam.abuobaida

[DESCRIPTION]
Introduction: Hepatitis B infection is a major public health problem worldwide, according to the WHO (World Health Organization) statistics in 2019 it affected 296 million people and over 6 million children under the age of five,It contributed to more than 820,000 death every year(1).According to its blood borne transmission nature of the virus, HBV (Hepatitis B virus) is very common in Khartoum and more likely to be endemic, a previous study which was conducted in Khartoum citizens, it prevailed that Hepatitis B Sero-prevalence among population varied from (47%) to (78%) which is very high and hepatitis B surface antigen prevalence ranging from (6.8%) in central Sudan to (26%) in southern Sudan(2).

 Objectives: The main aim of the study is to evaluate and assess the level of (KAP) toward (HBV) among (HCWs) in 60 Public and Private hospitals in Khartoum state, Sudan.
 Specific objectives are to estimate the proportion of immunized HCWs, to estimate the proportion of needle stick injuries and their relationships with the endemic status of HBV in Khartoum.

 Methods: A  Cross sectional descriptive study will be conducted. The study will be conducted in the capital of Sudan,Khartoum state including 60 public and private hospitals that are well distributed among the tripartite metropolis capital of Sudan. From late January 2023 to late March 2023.
 population size is (21,576) Samle size will be calculated using the proportion formula of a cross sectional study (n = required sample size n = Z (α/2) 2 pq/d2).
Size of population:(21,576), P value:(0.58), q(1-0.58)= (0.42) , (95%) confidence level and (2%) error margin.
Here, n = required sample size, n = Z (α/2) 2 pp./d2, p = 0.58. This resulted in the required sample size of 2111 HCWs.
A self administered questionnaire will be obtained from HCWs at the previously listed hospital.
Data will be analysed using SPSS version 28.

[STEPS]
SECTION: Research Steps
1. Writing the study protocol
SECTION: Research Steps
2.         Data collection and analysis                                    
SECTION: Research Steps
3. Writing the manuscript
SECTION: Research Steps
4. Publishing
TITLE: Acute Brain Slices 
AUTHORS: Beatriz E Nielsen

[DESCRIPTION]
This protocol describes the steps for preparing acute brain slices.

[BEFORE_START]
Prepare stock solutions: 10X Stock solutions can be made ahead of time and are shelf stable at Room temperature for 1 month; 1X solutions must be made fresh

10X ACSF

10X Cutting Solution


[STEPS]
SECTION: Acute Brain Slices
1. Set up vibratome and cutting station:
SECTION: Acute Brain Slices
1.3. Set up vibratome
Carefully clean blade with ethanol and water
Using a hex wrench, insert blade in blade holder assembly
Run VibroCheck calibration
Place buffer tray in ice tray and fill ice tray with ice; load on to vibratome
Superglue a rectangular piece (~2 cm x 1 cm x 1 cm) of 3% agar onto the specimen plate 
SECTION: Acute Brain Slices
1.1. Make 1X ACSF:
Prepare 1 L per mouse.
 900 mL + 100 mL  + 1.8 g  + 2 g 
Keep in water bath  32 °C  and bubble continuously with 95% O2 / 5% CO2.
Add ~30 mL to a slice holding vial, keep at 32 °C and bubble continuously with 95% O2 / 5% CO2.
SECTION: Acute Brain Slices
1.2. Make 1X cutting solution:
Prepare 250 mL per mouse
225 mL  + 25 mL + 0.5 mg  + 0.125 g +  4.3 g 
Chill on ice and bubble with 95% O2 / 5% CO2.

SECTION: Acute Brain Slices
1.4. Set up perfusion and cutting tools
SECTION: Acute Brain Slices
2. Perfuse animal with cold cutting solution
SECTION: Acute Brain Slices
3. Dissect brain and glue it to specimen plate.
SECTION: Acute Brain Slices
4. Insert plate in buffer tray and fill tray with 1X cutting solution.
SECTION: Acute Brain Slices
5. Lower blade into solution, set front and back limits and settings.
Slice at 0.8 mm/s
  

SECTION: Acute Brain Slices
6. Collect relevant slices as they come off the blade, and carefully transfer to warmed slice vial.
SECTION: Acute Brain Slices
7. Recover slices at 32 °C and bubbled for at least 45 minutes.
SECTION: Solution preparation
8. 10X Cutting solution stock


 


SECTION: Solution preparation
9. 10X ACSF stock
TITLE: No Title
AUTHORS: 

[BEFORE_START]
3-4 hour XZ-6E virus infected NC64A, pellets frozen at -80°C

[GUIDELINES]
<strong>MATERIALS AND ASSAY CONDITIONS:</strong><br /><br /> 1) 3-4 hour XZ-6E virus infected NC64A, pellets frozen at -80°C<br /> 2) Buffer A: <br />
<ul>
<li>10 mM Tris-HCl, pH 8.0</li>
<li>10 mM 2-ME</li>
<li>50 µg/mL PMSF</li>
</ul>
3) Buffer B: <br />
<ul>
<li>20 mM Tris-HCl, pH 7.8</li>
<li>0.5 mM EDTA</li>
<li>7 mM 2-ME, 10% Glycerol</li>
</ul>
4) Buffer C: <br />
<ul>
<li>10 mM KHPO<sub>4</sub>, pH 7.4</li>
<li>0.5 mM EDTA</li>
<li>7 mM 2-ME, 10% Glycerol</li>
</ul>
5) Storage buffer: <br />
<ul>
<li>20 mM Tris-HCl, pH 8.0</li>
<li>0.5 mM EDTA</li>
<li>0.1 mM DTT</li>
<li>50 mM KCl</li>
<li>5 mM MgCl<sub>2</sub>, 50% Glycerol</li>
</ul>
6) 4 M NaCl<br /> 7) 28% Polyethyleneglycol (PEG) 8000<br /> 8) Assay buffer and conditions:<br />       1X CviRI assay buffer:<br />
<ul>
<li>10 mM Tris-HCl, pH 7.8</li>
<li>150 mM KCl</li>
<li>10 mM MgCl2</li>
<li>100 µg/mL BSA</li>
</ul>
<br />All assays are carried out in 20.0 µL volumes with 1 µg of pUC19 DNA as substrate for 60 to120 min at 25°C. The assays are electrophoresed on 1.2% agarose gels (100 mL gels with double 20 lane combs) for 1 hour at 100 volts in 1X TPE buffer. Gels are stained with 0.5 µg/mL ethidium bromide for 30 min and visualized and photographed on a UV light box.

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Disturbing the rhythm of thought: speech pausing patterns in schizophrenia, with and without formal thought disorder
AUTHORS: Derya Cokal, Vitor Zimmerer, Douglas Turkington, Nicol Ferrier, Rosemary Varley, Stuart Watson, Wolfram Hinzen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Everyday speech is produced with an intricate timing pattern and rhythm. Speech units follow each other with short interleaving pauses, which can be either bridged by fillers (</span><span style = "font-style:italic;">erm</span><span>, </span><span style = "font-style:italic;">ah</span><span>) or empty. Through their syntactic positions, pauses connect to the thoughts expressed. We investigated whether disturbances of thought in schizophrenia are manifest in patterns at this level of linguistic organization, whether these are seen in first degree relatives (FDR) and how specific they are to formal thought disorder (FTD). Spontaneous speech from 15 participants without FTD (SZ-FTD), 15 with FTD (SZ+FTD), 15 FDRs and 15 neurotypical controls (NC) was obtained from a storytelling task and rated for pauses subclassified by syntactic position and duration. SZ-FTD produced significantly more unfilled pauses than NC in utterance-initial positions and before embedded clauses. Unfilled pauses occurring </span><span style = "font-style:italic;">within</span><span>clausal units did not distinguish any groups. SZ-FTD also differed from SZ+FTD in producing significantly more pauses before embedded clauses. SZ+FTD differed from NC and FDR only in ‘long’ (between 1 and 3 seconds) utterance-initial pauses. FDRs produced significantly fewer fillers than NC. Results reveal that the temporal organization of speech is an important window on disturbances of the thought process and how these relate to language. </span></div></div>

[STEPS]
TITLE: IPTG_induction
AUTHORS: Ana Belem García González, Georgina Diego, Jair Alexis Gardea Sáenz, Irán Alessandra Chaparro Rodríguez

[DESCRIPTION]
Protocol to induce protein expression using IPTG.

[STEPS]
SECTION: Protocol
1. From a relatively fresh plate, grab a colony and grow   at 37 °C and  in 10 mL of LB+CAM.

SECTION: Protocol
2. Inoculate 3 mL in 30 mL of LB+CAM and incubate at 37 °C  and  

SECTION: Protocol
3. When the cells reach to a OD600 between 0.4 and 0.8, the protein expression must be induced (IPTG concentrations can vary from 0.1 to 1M). 
SECTION: Protocol
4. Incubate a 37 °Cat  for 300 min (Hours can vary).
TITLE: UC Davis - Intraperitoneal Glucose Tolerance Test
AUTHORS: Fawaz G. Haj

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">
An intraperitoneal Glucose tolerance test or ipGTT is designed to determine clearance of an intraperitoneally injected glucose load from the body. Animals are fasted for approximately 16 hours, a solution of glucose is administered by intraperitoneal (IP) injection and blood glucose is measured at different time points during 2 hours post-injection.</div></div>

[STEPS]
?. Fast mice for 16hours by taking away food the day before (3:00pm)
?. The following day, Calibrate the glucose meter according to the manufacturer’s instructions.
?. Deprive mice from water then remove approximately 5 μl of blood (one drop) from thetail via a tail tip cut and transfer directly onto a glucose indicator strip.
?. Measure blood glucose immediately in a glucometer
?. Give the mouse an intraperitoneal injection of Glucose (2g/kg) with a 27 G needle
?. Continue to take blood samples from the initial tail cut before the glucose injection and at 15, 30, 60 and 120 min.
Between each of these time points, return the mouse to its cage and monitor it every minute.
?. NOTE:
At the end of the experiment, wipe tail with 70% alcohol and allow drying. Ensure that blood loss from the tail stopped before placing the animal back to its cage.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Moral licensing or, what is equivalently called, “self-licensing” is the use of a Good Act instrumentally: to cover up a Bad Act. This paper’s thesis is that moral licensing is inextricably the same as “instrumental apology,” i.e., bad-faith apology. For instance, a decision maker may issue an apology (Good Act) after committing a Bad Act. But if the decision maker uses the apology instrumentally, he or she is using the apology to justify the Bad Act and, hence, the apology is insincere. Sincerity is the fine line between good-faith apology or, more generally, Good Act, on one hand, and instrumental apology or, more generally, moral licensing, on the other. According to Kantian ethics, the sincerity behind an act, and not just the consequence, matters. This pits Kant against the utilitarian view, which downplays intentions, and focuses on consequences. We take Kant to the lab. Participants play a modified ultimatum game, where proposers in some treatments have the option to issue apology messages, and responders have both costly and costless options for rewarding or punishing. We introduce different treatments of the apology message to allow for responders to form doubt about the sincerity of the apology messages. Our results support the Kantian position: responders exhibit “insincerity aversion” by punishing proposers, when such punishment is costless to them, once they become suspicious of the sincerity of the apology of the proposers.

[STEPS]
TITLE: Pole test
AUTHORS: Pranay Srivastava

[DESCRIPTION]
Pole test is a behavior test for assessing motor dysfunction in rodents.

[STEPS]
1. Set up a vertical pole (1 cm diameter, 50 cm height) in a controlled testing
environment.
2. Allow the animals to acclimate to the testing environment for one hour before starting the test.
3. Mice were trained on the pole one day before the start of the experiment for 120s. Time taken by the mice to turn (T turn) and time taken to climb down (T descent) the pole were recorded (Matsuura et al. 1997; Sun et al. 2021).
TITLE: Virus Concentration and Infection
AUTHORS: Aditya Mohan

[STEPS]
?. [Lentivirus Concentration]
Harvest the lentivirus-containing supernatants. (Caution: supernatants contain live lentivirus.) Pool similar stocks, if desired. Filter through a 0.45 μm filter.
?. [RetroNectin Plate Preparation]
Prepare RetroNectin solution (30 ug/mL) by diluting RetroNectin powder (0.5 mg) into 16.6 mL of PBS
?. [Virus Infection]
Add the retrovirus stock solution or diluted solution at 125 - 500 μl/cm2 to theRetroNectin-coated plate. (Approx 1.5 mL)
?. [Lentivirus Concentration]
Transfer clarified supernatant to a sterile container and combine 1 volume of Lenti-X Concentrator with 3 volumes of clarified supernatant. Mix by gentle inversion. Larger volumes may be accommodated through the use of larger (i.e., 250 ml or 500 ml) centrifuge tubes.
?. [Lentivirus Concentration]
Incubate mixture at 4°C for 30 minutes to overnight.
4 °C
?. [Lentivirus Concentration]
Centrifuge sample at 1,500 x g for 45 minutes at 4°C. After centrifugation, an off-white pellet will be visible.
Centrifuge: 1500 34
4 °C
?. [RetroNectin Plate Preparation]
Dispense an appropriate volume of sterile RetroNectin solution into each well (1.5 mL) per 6 well dish.
?. [Lentivirus Concentration]
Carefully remove supernatant, taking care not to disturb the pellet. Residual supernatant can be removed with either a pipette tip or by brief centrifugation at 1,500 x g.
?. [Lentivirus Concentration]
Gently resuspend the pellet in 1/10 to 1/100th of the original volume using complete DMEM, PBS, or TNE. The pellet can be somewhat sticky at first but will go into suspension quickly.
?. [RetroNectin Plate Preparation]
Keep at room temperature for 30 minutes.
0 Room temperature
?. [RetroNectin Plate Preparation]
Remove the RetroNectin solution and then block with an appropriate volume of sterile2% bovine serum albumin (BSA, Fraction V) in PBS (1.5 mL of a 6 well dish) Allow the plate to stand at roomtemperature for 30 minutes.
0 Room temperature
?. [RetroNectin Plate Preparation]
Remove the BSA solution, and wash the plate once with an appropriate volume ofHBSS/Hepes or PBS. After removing the wash solution, the plate is ready for use.
?. [Virus Infection]
Place the plate in a centrifuge pre-warmed to 32℃ and centrifuge for2 hours at 32℃ at 1,000 - 2,000g to facilitate binding of virus particles withRetroNectin reagent.
32 °C
Centrifuge: 2000 34
?. [Virus Infection]
Discard the supernatant, but do not allow the plate to dry. Wash the plate with anappropriate volume of PBS or PBS containing 0.1 - 2% albumin (BSA or HSA).
?. [Virus Infection]
Collect the target cells and count the number of living cells. Then suspend the cellsin the growth medium at a concentration of 0.2 - 1 x 105 cells/ml.
?. [Virus Infection]
Do not allow the plate to dry. Immediately add target cells at a density of0.5 - 2.5 x 104 cells/cm2.* 6 well dish has SA of 9.6 cm2
?. [Virus Infection]
To promote contact between the target cells and viral particles, plates canbe centrifuged after adding the cells.    2000 g for 20 minutes
Centrifuge: 2000 34
?. [Virus Infection]
Incubate in a 37℃, 5% CO2 incubator for 2 - 3 days.
TITLE: Understanding the influence of altered lipid metabolism on TDP-43 pathology: a protocol for a systematic review and meta-analysis
AUTHORS: Holly Spence, Fergal M Waldron, Jenna Gregory, Tatiana Langerova

[DESCRIPTION]
Background: TDP-43 aggregation is the hallmark pathology seen at post-mortem in the majority of Amyotrophic lateral sclerosis (ALS) cases. Lipid metabolism has been shown to be disrupted in ALS including evidence for systemic metabolic disruptions in ALS patients as well as lipid peroxidation changes noted at the single cell level. The temporal nature of lipid dysregulation during disease course and the role that lipid dysregulation may play in potentiating TDP-43 pathology is unclear.

Aim: The aim of this review is to analyse existing literature assessing the interplay between altered lipid metabolism and TDP-43 pathology in ALS, and investigate possible relationships between the two.

Methods: This protocol describes the strategy for a proposed systematic review and meta-analysis to identify, evaluate and analyse studies examining lipid changes in relation to TDP-43 pathology.

Summary: This review will aid our understanding of ALS-associated pathobiology through detailed analysis of molecular processes underlying the influence of lipids on TDP-43 behaviour in ALS.

[STEPS]
TITLE: Yale - Tissue Glycogen
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure for determining the glycogen content within liver and muscle tissue.</div></div>

[STEPS]
?. Homogenization 1) Obtain samples of around 25mg. Keep samples in liquid nitrogen until ready to weigh. 2) Record weight. 3) Multiply weight of tissue by 5, and add that amount of 0.6N perchloric acid (PCA) to labeled 12x75mm plastic shaker tubes. Keep these tubes on ice. 4) Once done with above, add tissue to plastic homogenizing tubes and homogenize in shaker at 20Hz for 30 seconds. Do not throw out the PCA homogenized mixture until results are calculated.
?. Glycogen hydrolysis 5) Add 50µL of PCA homogenate to labeled microfuge tubes. 6) Add 25µL KHCO³ (1M) 7) Prepare fresh Amyloglucosidase solution (Amylogucosidase and Acetate Buffer in a 2mg enzyme /1mL buffer ratio. Pipette 125 µL of fresh amyloglucosidase into each microcentrifuge tube. 8) Incubate samples for 2 hours at 37ºC 9) Centrifuge for 1 minute at 10,000 rpm.
?. Background (free glucose in tissue homogenate) 10) While glycogen samples are incubating, centrifuge tubes containing PCA homogenate for 1 minute at 10,000 rpm. 11) Measure the glucose concentration of the PCA homogenate solution (Cobas Mira Glucose method) to obtain the amount of free glucose in each sample.
?. Glycogen content analysis12) Measure the amount of glucose (Cobas Mira Glucose method) in the 2hr-amyloglucosidase digested samples.
?. Glycogen Concentration Calculation13) Glycogen expressed as the gm % glycogen in tissue (i.e.,weight percentage of liver) is calculated as :
TITLE: Preparation for OCT embedding Mouse Kidney Tissue (TDA-University of Washington)
AUTHORS: kangsk, Runze Dong, Liangcai Gu

[DESCRIPTION]
Preparation for OCT embedding Mouse Kidney Tissue (TDA-University of Washington)

[STEPS]
SECTION: Kidney tissue collection
1. C57BL/6J male mice from the National Institute of Aging Rodent Resource were handled according to the guidelines of the Institutional Animal Care Committee at the University of Washington.  Mice aged 5 and 26 months were selected for the study. Kidney tissue extraction was conducted immediately following euthanasia.
SECTION: Kidney tissue collection
2. Make an incision then cut the skin from the bottom to the top of the mice followed the abdominal line with the round side of the scissors.
SECTION: Kidney tissue collection
3. Proceed to cut the lateral skin to get access to the kidney. Carefully extract the kidney, rinse it with HBSS (Hank's Balanced Salt Solution, Thermo, 14025092), remove the capsule, and section the kidney into pieces measuring 3 to 5 mm in thickness.
SECTION: OCT Embedding
4.  Apply a thin layer of OCT compound (Sakura Tissue-Tek O.C.T. Compound, Sigma, 4583) into a disposable mold (Epredia, 18985-1).
SECTION: OCT Embedding
5. Position the sample tissue at the center of the mold, ensuring that the side intended for cutting faces inward.
SECTION: OCT Embedding
6. Cover the tissue completely with additional OCT compound.
SECTION: OCT Embedding
7. Lower the mold into liquid nitrogen, being careful to not fully submerge the mold. Hold in liquid nitrogen for a few seconds until fully frozen.
SECTION: OCT Embedding
8. Transfer the frozen mold to dry ice for storage at -80ºC.
TITLE: Tail Immersion Test 
AUTHORS: Lani Tieu, Brent Boomhower, Olivier George

[DESCRIPTION]
The tail immersion test measures pain response (tail flick latency) to thermal stimuli, and can be used to evaluate the effectiveness of and tolerance to analgesics (e.g. oxycodone).

[BEFORE_START]
Set up water bath:
Fill approximately one-third of the Fisherbrand™ Isotemp™ GPD 02 Water Bath machine with water.
Fill beaker with approximately 900 mL water, then place beaker inside the water bath.
Keep a thermometer inside the beaker.
Turn the machine on (power switch located on the rear of the machine). The home screen will appear displaying the set point of the water temperature which should be 54°C.
Press the power button on the front of the machine to begin heating the bath. The screen will display the actual water temperature in the machine. 
Allow time for the temperature inside the beaker to reach 52°C before beginning experiment.
 

 
 


[GUIDELINES]
2 people (i.e. a dipper and a timer) are required to perform this test. Each timepoint that this test is performed must be done by the same people.

[STEPS]
SECTION: General Procedure
1. Pick up rat from its home cage and wrap it in a towel or pee pad to restrain it, leaving tail exposed.
SECTION: General Procedure
2. The dipper will hold the restrained animal over the water bath and count down before dipping 1 cm of the distal tail into the water. As soon as the tip of the tail is submerged, the timer will start the stopwatch to begin timing the rat's tail flick latency. 
 


SECTION: General Procedure
3. As soon as tail flick reflex is observed, the timer will stop the stopwatch and record the tail flick latency.
 

 

SECTION: General Procedure
4. Dry the rat's tail and return it back to its home cage.