eamag/protocols-with-steps huggingface dataset:
TITLE: Mouse synapse imaging and analysis
AUTHORS: Shiyi Wang

[DESCRIPTION]
Mouse synapse imaging and analysis

[STEPS]
1. **Tissue Sectioning and Preparation**
1.1. - Prepare coronal sections of 30 μm thickness containing the anterior cingulate cortex (ACC) and primary motor cortex (MOp) from WT and LRRK2 G2019Ski/ki mice.
2. **Synaptic Staining**
2.1. - Perform synaptic staining using the following antibody combinations:
2.2. - Excitatory (Intracortical): VGluT1 (pre-synaptic) and PSD95 (post-synaptic).
2.3. - Inhibitory: VGAT (pre-synaptic) and Gephyrin (post-synaptic).
3. **Antibody Incubation**
3.1. - Dilute primary antibodies (anti-VGluT1, anti-PSD95, anti-VGAT, anti-Gephyrin) appropriately in blocking solution.
3.2. - Incubate tissue sections overnight at 4°C with primary antibodies.
4. **Secondary Antibody Incubation**
4.1. - Wash sections with 1x TBS containing 0.2% Triton X-100 (TBST).
4.2. - Block with 10% normal goat serum (NGS) diluted in TBST.
4.3. - Incubate sections with Alexa Fluor-conjugated secondary antibodies (Life Technologies) for 2-3 hours at room temperature.
5. **Confocal Microscopy**
5.1. - Acquire high-magnification images using an Olympus FV 3000 inverted confocal microscope.
5.2. - Use a 60x objective with 1.64x optical zoom.
5.3. - Acquire z-stack images consisting of 15 optical sections spaced 0.34 μm apart.
6. **Image Processing**
6.1. - Convert each z-stack into 5 maximum projection images (MPI), each corresponding to a 1 μm section of the z-plane, using FIJI.
7. **Synaptic Puncta Analysis**
7.1. - Analyze synaptic puncta using FIJI plugin Puncta Analyzer153.
7.2. - Identify synapses by the colocalization of pre and postsynaptic puncta (VGluT1/PSD95 for excitatory synapses, VGAT/Gephyrin for inhibitory synapses).
8. **Data Collection**
8.1. - Analyze 15 MPIs per mouse (5 MPIs per tissue section, 3 tissue sections per mouse).
8.2. - Analyze between 4 and 5 age- and sex-matched mice per genotype and condition, as specified in the figure legends.
9. **Data Handling**
9.1. - Ensure all animals appear healthy at the time of collection.
9.2. - Include all data collected without exclusions, as per experimental design.
10. Notes:
11. - Maintain consistency in tissue processing and staining protocols.
12. - Perform all image analysis using standardized settings and procedures to ensure data reproducibility.
TITLE: Rotarod
AUTHORS: sdwalto, Bryan_Killinger, Jeffrey Kordower

[DESCRIPTION]
Rotarod protocol optimized for mice. This test is made to assess motor coordination and balance. The results should show lower latency to fall numbers for mice with motor impairments. 

[STEPS]
1. Training: Each mouse was given a training session. Four 5 min trials, 5 mins apart. 
2. Test Period (1 hour later): Each mouse was place inside the rotarod apparatus with increasing speed (4 rpm to 40 rpm in 300 secs). Latency to fall was recorded. (Digital rotarod recorded it automatically)
3. Each mouse received two consecutive trials. Mean latency to fall was used for analysis.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol describe step by step how to download and explore the dataset provided by the article Three-dimensional nanostructure of an intact microglia cell of Bolasco et al 2018.

[BEFORE_START]
In this protocol we describe how to navigate the dataset with Blender. Because it can be confusing at first, we recommend to follow a ten minute overview of Blender:  https://www.youtube.com/watch?v=kes2qmijy7w&list=PLa1F2ddGya_8V90Kd5eC5PeBjySbXWGK1 and https://www.youtube.com/watch?v=qCkHNxOf9lE&list=PLa1F2ddGya_8V90Kd5eC5PeBjySbXWGK1&index=2

Other software can be used to navigate the 3D model, but this won't be covered by this protocol.

[GUIDELINES]
This protocol have been tested only with a Windows 10 configuration. If you encounter any kind problem following the step by step, we strongly encourage you to provide feedback to improve this protocol.

[STEPS]
SECTION: Do the downloads and installs
?. 
SECTION: Link the addon to Blender
?. 
SECTION: Generate the images of the stack in X and Y orientation Full resolution (optional)
?. 
SECTION: Open the dataset in blender
?. 
SECTION: Generate the images of the stack in X and Y orientation Half resolution (recommended) (optional)
?. 
SECTION: Import the EM slices in Blender (optional)
?. 
SECTION: Add/remove object from the view, color and transparency
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Sabrina Geraci-Yee modified this protocol from: Pozarowski P., Darzynkiewicz Z. 2004. Analysis of cell cycle by flow cytometry. Methods Mol.<br />Bio. 281:301-311.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: tocsy_metab.nan
AUTHORS: NAN KB, John Glushka, Mario Uchimiya, Saraa Al Jawad, Christopher Esselman, Leandro I Ponce, Laura Morris, Arthur Edison

[DESCRIPTION]
This is a protocol for running the Bruker pulse program "dipsi2gpphpr" for metabolomics samples.

[BEFORE_START]
This protocol assumes:
Your sample is loaded, locked, tuned, and shimmed in the magnet
The calibrated 90° pulse value for proton (i.e., P1) for the sample has been collected

[GUIDELINES]
This protocol intends to provide concise instructions to carry out the experiment. For more comprehensive information, see Bruker's documentation "Basic NMR Experiments" by clicking ? →  Manuals (docs) on the menu bar on TopSpin. See also "Pulse Program Catalogue. 1D/2D" for the details about the pulse program used in this protocol.

[STEPS]
SECTION: Create a new dataset
1. 
SECTION: Create noesypr1d experiment file
0.23. When Lock, Tune, Shim are complete proceed to the specific protocol for your desired pulseprogram(s).
SECTION: Acquire a spectrum
2. Go to the "USE DEFAULT" tab below to proceed with the default optimized parameters.

SECTION: Create a new dataset
1.1. On the menu bar on TopSpin, click on
Start → Create Dataset
 

 

SECTION: Create a new dataset
1.2. Enter
NAME: Name of a set of datasets (e.g., human_serum_study1). Use a single string.
EXPNO: Dataset number. Use a positive integer.

Select
Directory: Your directory.
 

 

SECTION: Create a new dataset
1.3. Select
Read parameterset

Click the button
Select
SECTION: Create a new dataset
1.4. A new window opens. On the right top bar, select
Source = /opt/NAN_METAB/par
 

In the list, select the one you want to use:

For serum and plasma samples:
TOCSY_br600_serum.par: Parameter set optimized for serum samples.

For urine samples:
TOCSY_br600_urine.par: Parameter set optimized for urine samples.
TOCSY_NUS_br600_urine.par: Parameter set using an acquisition mode "non-uniform sampling (NUS)". Higher resolution on the indirect dimension

 

Click
OK
SECTION: Create a new dataset
1.5. Click
OK
3. 
3.1. Load the calibrated P1 using the following command in the command line.

 
(e.g., getprosol 1H 10.01 -7.45)

 

3.2. Click on
Acquire → Gain
in the menu bar to automatically set the receiver gain.
 

 

3.3. Click
Go
in the menu bar to acquire a spectrum.
 

3.4. After the run, click on
Process → Proc. Spectrum
in the menu bar to execute an automated processing macro.
 

3.5. If you want to modify parameters to improve your spectrum, go to step 2 and move to the step case "MODIFY PAR".
3. 
3.1. Select the AcquPars tab to show a parameter set.
 

 

3.2. Click the icon
_∏_
to see a complete parameter set.

 

3.3. Change parameters as needed.

Typically, the main parameter to adjust is NS:
 

3.4. Other potential parameters to adjust: ≥≥
 

3.5. Other potential parameter for the NUS parameter sets:
 

3.6. Acquire a spectrum. If more optimization is needed, go to step 3.3.
TITLE: Transform pCas9
AUTHORS: Qiaowa  Gong

[DESCRIPTION]
this protocol is aimed to provide instructions of transforming pCas9, which is a key step of knock-out gene by Crispr/Cas9

[STEPS]
1. Mix 50 µL of competent MG1655 with 10 µL of pCas9


2. Keep the reaction mix on ice for30 min 

3. Then, transfer the mix to the water bath set at 42 °C for 45 s 


4. Next, keep the mix on the ice for 2 min 

5. Add  940 µL LB broth and incubate the cells at 37 °C  with vigorous shaking (200 rpm) for 60 min 





6. Plate the cells on LB-agar plates containing chloramphenicol




7. Incubate at 37 °C
TITLE: Trim Clinical Keto Reviews – An Excellent Fat-Burning Ketosis Dietary Supplement!
AUTHORS: Trim Clinical Keto

[DESCRIPTION]
Trim Clinical Keto Reviews: Pills can make your weight reduction routine simpler than any time in recent memory! This effective recipe has the ability to drive your body into ketosis. What's more, ketosis is your body's head fat consuming interaction. Ordinarily, our bodies like to store the fat we eat. 

[STEPS]
TITLE: Ultra-High Macro Photography of Bark Beetles
AUTHORS: Demian F Gomez, Andrew J. Johnson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol explains the process of taking high quality photos of bark and ambrosia beetles, creating the image stacks, and processing the images in image editing software (Gimp or Photoshop).</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: Xing et al. 2016 Protocol
AUTHORS: Xing ey al., Ely Bor, Jade Knight

[DESCRIPTION]
Observation on tomatoes and green beans life spans when dipped in a chitosan spray and a combination of the chitosan spray with cinnoman oil. It is the replication of the "Chitosan-Based Coating with Antimicrobial Agents: Preparation, Property, Mechanism, and Application Effectiveness on Fruits and Vegetables" experiment by Xing et al. 2016.

[STEPS]
1. Prepared the CS solution to dissolving CS in an aqueous acetic acid solution at an ambient temperature overnight.
2. Added Tween 80 to the solution and Stirred at 45C for 2 hours
3. Dropped CEO in to the solution for 30 mins
4. Weight ratios of CS to CEO 1:1
5. Added drop-wise of TPP solution into o/w emulsion (for CEO-CSN) or CS solution (for CSN) under constant stirring at room temperature for 60 mins.
6. Nanoparticles were centrifuged at 27,000g for 14 mins
7. Prepared separately by dispersing 0.15% (w/v) of each one in distilled water and kept at 4 C for two coating solutions of CSN and CEO-CSN) with final concentration of 1.5 g/L.
8. Wash and dry tomatoes and green beans.
9. Separate the tomatoes by size: small, medium, and large. One of each size goes into one of the three groups. Separate the green beans by length: long and short. Split them equally between the three groups.
10. Separate the container into three sections and its lid into three sections. Label one "control" for tomatoes on the container and "control" for green beans on the lid. Lay the vegetables for one group in their corresponding sections.
11. Next label another section "CS" on both the lid and container. Dip the second group of vegetables into the Chitosan spray for 2 minutes each. Then lay them out in their sections.
12. Label the last section "CS+COE" like before. Take the last group and also dip all the vegetables for 2 minutes each and lay them out in their sections.
13. Every three days observe and changes and take a photo. Repeat until 15 days are reached.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This questionnaire serves to interview subjects undergoing rehabilitation of joint movement by means of exoskeleton robot HAL (hybrind assistive limbs). The goal is to learn about subjective control strategies during voluntary initiation of joint movement. Questions and multiple-choice answers build on phenomenological introspection. The results of the questionnaire are correlated to a gait score analysis.</p>

[GUIDELINES]
<div>
<p>The questionnaire might cause mental burden for the subjects because they might recall their experience of not being able to move. This could raise traumatic experiences and result in severe mental stress. This risk has to be minimized by carefully selecting subjects that are mentally and emotionally stable enough to face their experience of impairment. Therefore, the selection of subjects will be done based on the judgment of a medical doctor.</p>
<p>Furthermore, the workload of the study is minimized by conducting the questionnaire within one hour (including the preparation).  In case that the subjects do not feel well or need to lay down due to their physical conditions, subjects have the possibility to take a rest any time during the interview. During the interview, the interviewer has to carefully observe the subject and stop the questionnaire in case of any signs of stress on the side of the subject. Medical staff has to be available.<br /><br />In order to decrease the subjects’ overall burden, the interview is to be conducted just after finishing the last HAL session.</p>
<p> </p>
<p>In order to avoid any burden on the subject's locomotor system, subjects should keep sitting on a seat during answering the questionnaire so that they do not need to move or engage in any physical exercise.</p>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Intracardial perfusion of the African turquoise killifish
AUTHORS: Valerie  Mariën, Jolien    Van houcke, Lutgarde  Arckens

[DESCRIPTION]
This perfusion protocol is essential for preserving tissue morphology in order to perform good quality immunohistochemical stainings. Here, we show you how we perform our perfusions on the African turquoise killifish.

This protocol was already used in the following publications:
Aging impairs the essential contributions of non-glial progenitors to neurorepair in the dorsal telencephalon of the Killifish Nothobranchius furzeri - PubMed (nih.gov)
Single-cell sequencing of the adult killifish (N. furzeri) brain identifies an atypical progenitor, glial and neuronal heterogeneity | bioRxiv


[STEPS]
SECTION: Preparation
1. Prepare 0.1% tricaine solution in reverse osmosis water.
Prepare 4% paraformaldehyde (PFA) solution in PBS.
Disinfect all dissection material with 70 % EtOH.
Cut the needle off the Terumo Needle Surflo Winged infusion set.
  

SECTION: Preparation
2. Fill one of the 10 ml syringes with PBS and the other one with 4% PFA. 
Attach the Terumo Surflo tube onto the syringe and fill the tube.
Open the tip of the capillary with forceps and attach the capillary onto the Terumo Surflo tube. Push out the fluid until a drop is visible at the end of the capillary.
  
  

SECTION: Preparation
3. Euthanize a fish in an overdose (0.1% ) Tricaine solution. After 10 minutes, the fish is euthanized.
SECTION: Start dissection
4. When the fish is euthanized, place the fish on the paraffin tray with its belly pointing up. 
Hold the head with forceps while gently making a T-shaped incision. Start cutting from the cloaca to the left and right dorsal sides. 
Be careful not to cut into any organs!
Start cutting towards the head from the opening that was just made parallel to the body. 
These steps can be carried out by using the bigger scissors (Item No. 15018-10).
Pin down the loose parts on the sides with pins or needles.
Use the small scissors to cut up till the gills in order to expose the chest cavity. Also pin down the sides close to the head. The heart is now visible and contracting.
 


SECTION: Start Perfusion
6. The fish is now perfused and you can collect your tissue of interest for further processing. For example collect the brain and let it post-fix overnight in a 4% PFA solution at 4°C in a 24 well-plate.


SECTION: Start Perfusion
5. Cut in the atrium to allow blood to flow out. 
Insert the capillary connected to the syringe containing PBS in the ventricle of the heart and gently push PBS through the heart and into the vascular system. You can see that the ventricle will swell up. The liver will become whiter as blood is flowing out the atrium of the heart. 
When the liver is white, most of the blood is removed from the body (approx. 5 minutes of flushing with PBS). Then, insert the capillary connected to the syringe containing PFA into the ventricle of the heart and let the PFA flow through the vascular system for another 4-5 minutes.
TITLE: SPARC Long-term exposure to intermittent hypoxia (or normoxia) using a custom in-cage computer controlled system
AUTHORS: Elisa Gonzalez-Rothi, Latoya Allen, Alec Simon, Yasin Seven, Marissa Ciesla, Gordon Mitchell

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedures for conducting long-term exposure to various intermittent hypoxia (or normoxia) protocols in rats. </div></div>

[STEPS]
?. Rats are exposed to either long-term normoxia or IH treatments (various protocols) for up to 28 days
?. Rats are housed in custom-designed PlexiGlas gas exposure cages (Volume~16L) with free access to food and water
?. Cages are fitted with a custom cage top equipped to deliver protocol-specific gas (O2 balanced with N2) in specified cycle intervals via programmable mass-flow controller (Therapeutiq, Madison, WI).
?. Exposure Protocols Include: Normoxia (Nx), daily acute intermittent hypoxia (dAIH), chronic intermittent hypoxia simulating mild sleep apnea (apnea-hypopnea equivalent = 6 episdoes/hour) (IH-5/5), or chronic intermittent hypoxia simulating moderate sleep apnea (apnea-hypopnea equivalent = 15 episdoes/hour) (IH-2/2)
?. Cages are filled with low-dust emitting bedding (Tek-Fresh) and are changed 3 times per week
?. Rats and gas exposure system are monitored at least 1x/hour during daily exposure to ensure system functionality and rat health/wellbeing
?. Gas flow rate through cages is set to 16 L/min (~60 air changes per hour) to prevent CO2 and ammonia buildup
?. All gases are medical grade and are filtered for small microparticles and bacteria using a high purity compressed air filter (Parker Hannifin)
?. Filtered air is humidified to reach a relative humidity of ~30-50% in the final gas mixture. The humidification system is comprised of a Nafion Fuel Cell Humidifier (Perma Pure, Lakewood, NJ) connected to the filtered air line and a water bath.
?. Normoxia Exposure:
?. Continuous gas flow of 21% O2 for 8 hrs/day
?. Daily Acute Intermittent Hypoxia Exposure (dAIH)
?. 10, 5-minute episodes of 10.5% O2 alternating with 5 minute normoxic intervals (21% O2) for a total duration of 1.5 hours/day (10 hypoxic episodes per day)
?. Chronic intermittent hypoxia simulating mild sleep apnea (IH-5/5): this protocol results in 6 hypoxic episdoes per hour which is equivalent to the clinical criteria (apnea-hypopnea index, AHI) for diagnosing mild sleep apnea
?. 5-minute episodes of 10.5% O2 alternating with 5 minute normoxic intervals (21% O2) for a total duration of 8 hours/day (6 hypoxic episodes per hour for 8 hours = 48 hypoxic episodes per day)
?. Chronic intermittent hypoxia simulating moderate sleep apnea (IH-2/2): this protocol results in 15 hypoxic episdoes per hour which is equivalent to the clinical criteria (apnea-hypopnea index, AHI) for diagnosing moderate sleep apnea
?. 2-minute episodes of 10.5% O2 alternating with 2 minute normoxic intervals (21% O2) for a total duration of 8 hours/day (15 hypoxic episodes per hour for 8 hours = 120 hypoxic episodes per day)
TITLE: Levantamento em bases de dados para Revisões Sistemáticas e Quantitativas da Literatura (RSQL)
AUTHORS: Daniel  Vartanian

[DESCRIPTION]
Este protocolo foi criado para o programa de Revisões Sistemáticas e Quantitativas da Literatura (RSQL) científica do Grupo Interdisciplinar de Pesquisa em Sono (GIPSO), mas ele pode ser utilizado por qualquer um que queira realizar uma RSQL. O programa foi criado para que novos ingressantes do GIPSO possam dar um pontapé inicial em uma carreira acadêmica na cronobiologia. Saiba mais no protocolo abaixo.
 
Para dúvidas ou sugestões, entre em contato em gipso@usp.br.

[BEFORE_START]
Veja o vídeo de instrução abaixo.

  
Tenha à mão os seguintes materiais antes de iniciar o protocolo:

Computador.
Última versão do aplicativo de navegação na web (aka browser) Chrome ou Firefox instalado no computador.
VPN (Virtual Private Network) USP configurado e ativado no computador (confira este tutorial da SIBiUSP para saber mais).
Aplicativo de compactação de arquivos 7-zip (para Windows) ou Keka (para macOS) instalado no computador.
Acesso à internet estável.

ATENÇÃO

Alguns provedores podem requerer procedimentos que não estão listados aqui. Consulte seu supervisor caso tenha dúvidas.
Alguns provedores só podem ser acessados utilizando o VPN USP. Certifique-se que você esteja usando ele antes de iniciar a busca.

[STEPS]
SECTION: Preparação
1. Acesse o VPN da USP
 

SECTION: Preparação
2. Abra as planilhas Source e Search
 

SECTION: Preparação
3. Preencha os dados iniciais na planilha Search
 

SECTION: Busca
4. Acesse o provedor da(s) base(s) de dados
 


SECTION: Busca
5. Selecione somente as bases de dados que irá utilizar (se aplicável)
 

SECTION: Busca
6. Limpe o histórico de busca
 

SECTION: Busca
7. Localize o campo destinado à busca avançada e desative possíveis filtros (se aplicável)
 

SECTION: Busca
8. Pesquise set por set, começando pelo primeiro da lista

 

SECTION: Registro
9. Salve o histórico de busca
 

SECTION: Registro
10. Faça o upload do histórico de busca no Google Drive
 

SECTION: Registro
11. Preencha a planilha Search com o link para o histórico de busca e com os resultados das pesquisas de cada set
 


SECTION: Registro
12. Acesse o set de interesse e realize o teste de aderência
 

SECTION: Registro
13. Preencha a planilha Search com o resultado do teste de aderência
 

SECTION: Extração
14. Exporte todas as referências encontradas pelo set de interesse
 

SECTION: Extração
15. Compacte os arquivos das referências em um arquivo no formato ZIP
 

SECTION: Extração
16. Faça o upload das referências compactadas para o Google Drive
 

SECTION: Extração
17. Preencha a planilha Search com o link para o arquivo das referências compactadas e com a data/hora final da busca
 

SECTION: Finalização
18. Adicione comentários a respeito da busca na planilha Search (se aplicável)
 

SECTION: Finalização
19. Delete os arquivos baixados no seu computador
 

SECTION: Finalização
20. Feche as planilhas e desconecte o VPN USP
 

SECTION: Finalização
21. Cheque o item do provedor e idioma selecionado no issue da atividade no GitHub
TITLE: Video protocol for sorting Drosophila pupae
AUTHORS: Carolyn Elya

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is my favorite way to sort pupae.</div><div class = "text-block">Credit to Tom Alisch & Dave Zucker (@FlySorter) for discovering a new use for transparencies!!</div></div>

[STEPS]
?. Video summary of the method:
?. Cut out a small piece of a projector sheet (e.g. Hygloss Overhead Projector Sheets, https://www.amazon.com/Hygloss-75910-Products-Transparency-Projectors/dp/B077762QK5/ref=sr_1_8?dchild=1&keywords=transparency&qid=1605473049&sr=8-8). You'll want to make sure the piece is long enough to completely cover the inside of the vial and not so wide as to reach beyond the surface of the food. Measure diameter of the vial mouth to determine a suitable length (d*pi + ~ 1 cm) and space between food and cap to determine width.
?. Roll up the projector sheet and place into a vial any time before flies reach 3rd instar.
?. Pull out the transparency after pupation.
?. Sort pupae using a paintbrush, using water to dissolve the glue holding pupae to the projector sheet. Placing a Kimwipe under the projector sheet will help you maintain a clean workspace and prevent inter-vial contamination.
?. Between genotypes, check paintbrush bristles for any adhered larvae or pupae and remove before proceeding. Rinse paintbrush thoroughly with water and 70% ethanol to decontaminate.
?. Pupae can be directly transferred to a new vial with food or placed on a new piece of projector sheet which can then be rolled up and placed into a vial.