eamag/protocols-with-steps huggingface dataset:
TITLE: Chemicals and treatment paradigms
AUTHORS: Pranay Srivastava

[DESCRIPTION]
This protocol is to enhance microglia response and nigral dopaminergic cell death in an acute MPTP model following peripheral inflammation resulting from single i.p. injection of LPS at 2 mg/kg (García-Domínguez et al. 2018).

[STEPS]
1. Mice were randomly divided into MPTP+LPS+NDP-MSH, MPTP+LPS, and control groups to receive i.p. once daily MPTP.HCl (Millipore Sigma, Cat# M0896; 20 mg/kg) or saline and LPS (Millipore Sigma, Cat# L4391; 1 mg/kg) or PBS from day 1 to day 4. 
2.  NDP-MSH (Genscript, Cat# RP10658; 400 µg/kg) or PBS was injected from day 1 to day 12. 
3. Mice were tested for behavioral activities and were sacrificed thereafter on day 12. 
SECTION: To study the role of Tregs
6. Another dose of anti-mouse CD25 monoclonal antibody or isotype control was administered 2 days before the sacrifice. 
SECTION: To study the role of Tregs
4. Animals were treated with anti-mouse CD25 monoclonal antibody (clone PC61, Biolegend, Cat# 102059; 400 µg/mouse) or isotype control (Biolegend, Cat# 401916; 100µg/mice) for 3 alternate days, 1 week before the start of experiment.
SECTION: To study the role of Tregs
5. Mice were subsequently treated with MPTP, LPS and NDP-MSH as described above.
TITLE: Primary microglial culture
AUTHORS: Xiqun Chen, Qing Ye

[DESCRIPTION]
Mixed glial cells were obtained from C57BL/6 mice embryonic day 17. Cells were cultured in high-glucose DMEM/F12 supplemented with 10% FBS in humidified air containing 5% CO2 at 37°C. The culture medium was replaced with fresh medium 24 h after the initial preparation and every 3 days thereafter. After 1 week, microglial cells were obtained by mechanical shaking of the mixed glial cell cultures for 1 h. Cells were routinely monitored for purity by ionized calcium-binding adaptor molecule 1 (Iba1) staining and the population of Iba1+ cells was >95% 

[STEPS]
1. Primary microglial culture  - Use C57BL/6J mice at embryonic day 17 
2. Anesthetized pregnant mice (1% sodium pentobarbital, 80mg/kg), dissect their embryos and collect the cortex. 
(Separate and remove the soft membrane and blood vessels, rinse the cerebral cortex in PBS, and use the ophthalmic scissor to cut pieces of the cortex)
4. Transfer the cortices to 15 ml tubes containing 1.5 ml trypsin–EDTA (0.25%) and incubate it at 37 °C for 15 min Dissociate the cortices by triturating with a 10 mL serological pipette 10 – 15 times
7. Change the culture medium the next day followed by the addition of a fresh culture medium every 10080 min till 14 days of culture 

8. Put the flask on a  shaker for 360 min at 37 °C  (The microglia grow as a monolayer on the top)
Collect the detached cells, centrifuge (400 x g, 5 min) and resuspended in 4 mL of  

Count the cells and plate at a density of 50,000 cells/cm2 in a 24 well plate 
Incubate them at 37 °C in 5% CO2. After 120 min microglia usually attaches to the bottom of the plate.  

The purity of the cells was monitored by staining with ionized calcium-binding adaptor molecule 1 (Iba1) antibody
(Microglia obtained by this method have a purity of 90%-95% and can be identified by immunofluorescence staining. 
9. Transduction with BRAF (Optional)

The purified microglial cells were seeded at a density of 80% and transduced with BRAFV600E, or BRAFWT, or vector lentivirus with 8 μg/ml polybrene (Sigma–Aldrich, USA) for 1440 min.

10. After transduction, the cells were cultured in  with or without  for 7200 min , and the medium, as well as the cells, were collected for subsequent experiments.


3. Collect the cortices in PBS  in a 50 ml tube on ice
(The 50 ml tube contains 30 ml of PBS) on ice 
6. Count the cells and plate them in a density of 50,000 cells/cm2 into PLL coated T-75 flask. Makeup the volume to 15 ml with the  
5. Centrifuge the dissociated cortices (400 x g, 5 min). Aspirate the media and resuspend the pellet in 5 ml
TITLE: Coimmunoprecipitation
AUTHORS: Shiyi Wang

[DESCRIPTION]
Co-Immunoprecipitation assays in HEK cells

[STEPS]
1. **Transfect HEK293T Cells** - Transfect HEK293T cells with Ezrin/Atg7 cDNA using X-tremeGENE HP (Roche) 36 hours prior to lysis. - Grow cells to 85-90% confluency.
2. **Collect Cells for Lysis** - At 36 hours post-transfection, rinse cells with 1x PBS. - Collect cells for lysis.
3. **Lysis and Protein Extraction** - Briefly vortex cells in chilled membrane solubilization buffer (25 mM HEPES, 150 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 10% Glycerol) with protease (cOmplete, Roche) and phosphatase (PhosSTOP, Roche) inhibitors.
4. **Equalize Protein Concentrations** - Use the Pierce™ BSA Protein Assay Kit (Thermo Fisher) and CLARIOstar Plus Plate Reader (BMG Labtech) to determine protein concentrations. - Equalize cell lysate concentrations.
5. **Incubate with Magnetic Beads** - Incubate equalized cell lysates with Pierce™ Anti-c-Myc Magnetic Beads (Thermo Fisher) for 6 hours at 4°C while rotating.
6. **Wash Magnetic Beads** - Wash beads with chilled lysis buffer.
7. **Elute Protein Samples** - Elute protein samples from beads by adding 2x Bromophenol Blue-free Laemmli Sample Buffer. - Heat samples to 95°C for 5 minutes.
8. **Perform Western Blot Analysis** - Subject eluted protein samples to western blot analysis using a SimpleWestern Jess (ProteinSimple) automated immunoassay system with a 12-230 kDa Fluorescence Separation module and the manufacturer's protocol.
9. **Primary Antibodies for Detection** - Use the following primary antibodies: - Anti-Myc (Rabbit, 1:40, Cell Signaling Technologies, mAb#2278) - Anti-HA (Rat, 1:20, Roche, 11867423001)
10. **Secondary Antibodies for Signal Detection** - Use the following fluorescently conjugated secondary antibodies at a 1:100 dilution: - IRDye® 680RD Goat anti-Rabbit IgG (LI-COR, 926-68071) - IRDye® 680RD Goat anti-Rat IgG (LI-COR, 926-68076)
11. **Quantify Protein Signals** - Quantify protein signals using the Simple Western Compass software. - Utilize quantitative electropherograms of detected signals. - Background-subtract and normalize Ezrin co-immunoprecipitation signal intensity to Ezrin protein load and Atg7 IP levels for statistical analysis.
12. **Statistical Analysis** - Perform statistical analysis in GraphPad Prism 9. - Use One-way ANOVA with Tukey’s multiple comparisons test. - Set alpha threshold to 0.05.
TITLE: High Resolution Imaging Mass Spectrometry Analysis using Bruker Daltonics Platforms
AUTHORS: Elizabeth Neumann, Jamie Allen, David Anderson, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Scope:</div><div class = "text-block"><span>Acquire 10 μm spatial resolution imaging mass spectrometry (IMS) datasets of ~ 5mm</span><span style = "vertical-align:super;">2  </span><span>regions of tissue.</span></div><div class = "text-block">Expected Outcome:</div><div class = "text-block">A series of ion images that can be used to visualize the distribution of lipids localizing to physiological regions within the human kidney.</div></div>

[STEPS]
?. Scann a 3200 dpi image of the matrix coated tissue section in the MTP slide adapter 2 with sufficient contrast to visualize the tissue boundaries.
?. Choose a method that has "height correct" initiated upon sample docking.
?. While the height correct profile is being generated, open FlexImaging and follow the prompts to designate sample file name, method, pitch, scanned image of the slide, etc.
?. Once the height correct profile has been generated, train the target position with three teaching points following the instructions within FlexImaging.
?. Draw a measurement region around the tissue for IMS analysis.
?. Go to an area off of the tissue and ensure the focus of the laser is adequate for 10 μm spatial resolution imaging.
?. Start the acquisition.
?. Insert the slide adapter plate into the instrument airlock.  Then press "load."
TITLE: Phage Buffer
AUTHORS: Dr. Steven Wilhelm

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

[STEPS]
?. In a one liter bottle, add 10 ml MgSO4.
[1 Molar]
?. Add 1 ml CaCl2
[1 Molar]
?. Add 1 ml Tris-HCl (pH~7)
[1 Molar]
?. Add 1 g gelatin.
1 g
?. Add Milli-Q water to a final volume of 1000 mL and autoclave sterilize.
TITLE: qPCR of α-synuclein, TNF and NF-κβ 
AUTHORS: Michael J Hurley

[DESCRIPTION]
A procedure for quantitative real time reverse transcription PCR of α-synuclein, TNF and NF-κβ 

[STEPS]
SECTION: qPCR
1. Extract total RNA and protein from cells or tissue using TRI Reagent® solution according to the manufacturer's instructions. Use RNase free glycogen as a carrier for the total RNA.
Keep protein for use in western blot.

SECTION: qPCR
3. Measure the concentration of RNA with a spectrophotometer (e.g. Nanodrop 1000). 
SECTION: qPCR
4. Make cDNA from 2 micrograms of total RNA with a high-capacity cDNA reverse transcription kit (Invitrogen) according to manufacturer's instructions.
SECTION: qPCR
5. Dilute cDNA from the reverse transcription reaction 1:10 with nuclease free water.
SECTION: qPCR
6. Amplify 2 microliters of cDNA with gene specific intron-spanning primers (see materials) using PowerUp™ SYBR™ Green Master Mix (Invitrogen) according to the manufacturer's instructions with a StepOne Real-Time PCR system (Applied Biosciences).
SECTION: qPCR
7. Analyse using the using the comparative 2-ΔΔCT method. 
SECTION: qPCR
2. Dissolve the RNA in nuclease free water.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Blue/White Screening of Bacterial Colonies X-Gal/IPTG Plates

[BEFORE_START]
Prepare 20 mg/ml X-Gal solution in DMF (See X-Gal Stock Solution Procedure). For reduced DMF toxicity in media, you can alternatively make a 100 mg/ml X-Gal solution in DMF (this concentration is only stable at -20&deg;C for ~1 week).<br /><br />Prepare 100mM IPTG solution in dH2O (or dilute from 1M IPTG Stock Solution).

[GUIDELINES]
Dry X-Gal/IPTG-coated media in a laminar flow hood for approximately 30 minutes before use.<br /><br />Plate Surface<br />1. Dry media plates in a laminar flow hood.&nbsp;<br />2. Add 40 &micro;l 100mM IPTG and 120 &micro;l X-Gal (20 mg/ml) to the surface of each plate and spread over&nbsp;entire surface.<br />Note: The edges of the plate are difficult to spread adequately and may give false positives. We advise picking colonies in the middle of the plate, if possible, for best results.

[STEPS]
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TITLE: Sarah-Blessing-CBD-Gummy-Fr
AUTHORS: health  

[DESCRIPTION]
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[STEPS]
1. The Experts’ Guide To Sarah Blessing CBD Gummies Fr

2.
TITLE: Whole colony fixation
AUTHORS: Simon Blanchoud

[STEPS]
?. [Anesthesia ]
Dilute   2x ASW with  dH2O in a Falcon tube.
25 mL
25 mL
?. [Anesthesia ]
Transfer the colony to the filled Falcon.
?. [Anesthesia ]
Add menthol crystals to the ASW and close the container.
?. [Anesthesia ]
Leave the colony to anesthetize for  .
?. [Anesthesia ]
Shake the 50x eugenol stock hard to mix the oil well, it should turn cloudy.
?. [Anesthesia ]
Add  50x eugenol to the colony's water and close the container.
1 mL
?. [Anesthesia ]
Leave until total relaxation of the colony's siphons; around  .
?. [Fixative]
Heat  dH2O in a beaker at   using a microwave.
25 mL
60 °C
?. [Fixative]
Place the beaker on a stirring hot   plate in a fume hood.
60 °C
?. [Fixative]
Add  of PFA (4 % final concentration) powder to the stirring water.
2 g
?. [Fixative]
Wait for the powder to sink in the water (increase stirring if needed).
?. [Fixative]
Add  NaOH drop by drop until the solution clears.
?. [Fixative]
Add  HCl drop by drop until solution reaches pH 7.5-8.2.
?. [Fixative]
Add  2x ASW and cool at  .
25 mL
0 Room temperature
?. [Fixation]
Mix  4 % PFA with  50X eugenol in a Falcon tube.
50 mL
1 mL
?. [Fixation]
Transfer the anesthetized colony to the fixative.
?. [Fixation]
Colony should be fully relaxed, if not extend anesthesia or increase eugenol.
?. [Fixation]
Fix  over night at
0 Room temperature
?. [Fixation]
Rinse in 1x ASW.
?. [Fixation]
Wash twice  in 1x ASW at   (under rocking if available).
0 Room temperature
?. [Fixation]
Wash three times  in PBS (under rocking if available).
?. [Fixation]
Detach the colony from the slide using a sharp razor blade.
?. [Fixation]
Keep in PBS at  .
4 °C
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol, based on published work, demonstrates how to delivery Cas9 RNP-based gene editing reagents to cultured mamallian cells by electroporation with a Lonza 4d Nucleofector. Consider consulting some of the following papers:</p>
<p> </p>
<p>1. RNP delivery paper upon which this work is based (Open Access):</p>
<p>https://elifesciences.org/content/3/e04766</p>
<p>2. Paper by an IGI post-doc that details the rationale behind HDR donor design:</p>
<p>https://www.ncbi.nlm.nih.gov/pubmed/26789497</p>

[BEFORE_START]
<p>You will need the following materials:</p>
<p> </p>
<p>1. Purified Cas9 NLS protein, 40 µM</p>
<p>2. Purified sgRNA from in vitro transcription, &gt;25 µM</p>
<p>3. single-stranded DNA HDR donor, 100 µM (as an IDT Ultramer)</p>
<p>4. Lonza 4d Nucleofector with X Unit</p>
<p>5. Lonza 'S' kit: electroporation solution and 16 reaction small-sized cuvettes</p>

[STEPS]
?. 
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TITLE: Diaétoxile : mise à jour, avis, prix officiel ici
AUTHORS: health  

[DESCRIPTION]
Diaetoxile est un produit naturel qui n'a pas d'effets secondaires. Lisez à propos des arnaques, des avis sur les prix et où trouver.

[STEPS]
1. Avis sur les Diaetoxile  : Les problèmes de surpoids et d'obésité sont devenus très courants dans la vie de nombreuses personnes. Les gens n'ont aucun contrôle sur leurs habitudes alimentaires. Consommer de la malbouffe et de la restauration rapide peut entraîner différents types de problèmes de santé. Les personnes qui consomment beaucoup d'aliments gras grossissent très rapidement. C'est parce qu'une grande quantité de graisse brune est stockée dans leur corps. Les niveaux de cholestérol augmentent également en raison de la consommation d'aliments malsains. Le poids des personnes augmente lorsqu'elles consomment beaucoup de nourriture et ne les brûlent pas. Afin de brûler la quantité de nourriture consommée, il est très important de faire bouger le corps. Consommer beaucoup de nourriture et ne faire aucun mouvement ou exercice entraîne un stockage des graisses. Beaucoup de gens, même après avoir fait des exercices, grossissent. C'est à cause de leur taux métabolique lent. Le taux lent du métabolisme stocke plus de graisse dans le corps par rapport à un taux métabolique rapide. Les personnes qui ont un taux métabolique rapide brûlent facilement les graisses consommées et restent en forme et en bonne santé. De nos jours, de faux produits chimiques et conservateurs sont ajoutés aux aliments que nous consommons. Diaetoxyl Même si nous consommons la nourriture que nous cuisinons, cette nourriture n'est pas non plus pure. La nourriture disponible sur le marché contient beaucoup de conservateurs ajoutés. L'utilisation de Diaetoxile peut vous aider à brûler les graisses en un temps record. 
Site officiel : https://ipsnews.net/business/2022/07/05/diaetoxile-avis-france-2022-detoxil-600-mg-en-pharmacie-prix-medical-negatif-forum-acheter-en-france/ ,  https://ipsnews.net/business/2022/07/06/diaetoxyl-avis-france-2022-diaetoxile-600mg-gelules-prix-en-pharmacie-medical-ou-acheter-en-france/
TITLE: Optimal Max Keto Reviews Shark tank pills scam or legit?
AUTHORS: optimalmaxetoreviews  

[DESCRIPTION]
Optimal Max Keto Reviews Shark tank pills scam or legit?


[STEPS]
1. Optimal Max Keto Reviews Shark tank pills scam or legit?
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TITLE: UroMOCA and StimPod Daily Testing in Pig
AUTHORS: Dennis Bourbeau, Brett Hanzlicek

[DESCRIPTION]
This protocol is for the daily testing of StimPods implanted in a pig.  Data from the StimPods and UroMOCA are recorded during these sessions.

[STEPS]
SECTION: Tolerance Testing
1. Attach BluMOCA receiver/antenna to harness/jacket
SECTION: Tolerance Testing
2. Charge StimPods, then confirm wireless connection to computer
SECTION: Tolerance Testing
3. Wake up UroMOCA
SECTION: Tolerance Testing
4. Stimulation permutations

SECTION: Tolerance Testing
4.1. 20 Hz, 500 Hz, 10 kHz
SECTION: Tolerance Testing
4.2. Stim0, Stim1
SECTION: Tolerance Testing
5. Stimulation duration: 1-5 seconds ON, >30 s rest
SECTION: Tolerance Testing
6. For each stimulation permutation, determine upper limit of stimulus intensity/amplitude without distress or discomfort to animal
SECTION: Tolerance Testing
7. Document subject’s responses   
SECTION: Recruitment Curve
8. Attach BluMOCA receiver/antenna to harness/jacket
SECTION: Recruitment Curve
9. Charge StimPods, then confirm wireless connection to computer
SECTION: Recruitment Curve
10. Wake up UroMOCA
SECTION: Recruitment Curve
11. Administer stimulation while recording bladder pressure and EMG
SECTION: Recruitment Curve
11.1. Stimulus duration: 5 seconds on, > 30 s rest
SECTION: Recruitment Curve
11.2. Stimulus amplitude range: [0:25 µA:tolerance limit]
SECTION: Recruitment Curve
11.3. Stimulus amplitudes randomized within range
SECTION: Recruitment Curve
11.4. For each stimulation permutation
SECTION: Recruitment Curve
11.5. Within tolerance range for that permutation
SECTION: Recruitment Curve
12. Document subject’s responses   
SECTION: Simultaneous Stimulation
13. Attach BluMOCA receiver/antenna to harness/jacket
SECTION: Simultaneous Stimulation
14. Charge StimPods, then confirm wireless connection to computer
SECTION: Simultaneous Stimulation
15. Wake up UroMOCA
SECTION: Simultaneous Stimulation
16. Stimulus duration: 5-10 seconds on
SECTION: Simultaneous Stimulation
17. Amplitude for 20 Hz stimulation: 90% of full recruitment
SECTION: Simultaneous Stimulation
18. Amplitude for 500 Hz or 10 kHz stimulation: minimum amplitude (threshold) to achieve maximum EMG reduction
SECTION: Simultaneous Stimulation
19. Measure bladder pressure and EMG
SECTION: Simultaneous Stimulation
20. Document subject’s responses
TITLE: Scanning Electron Microscopy - Focused Ion Beam Biological Sample Preparation
AUTHORS: Ludovica   Parisi

[DESCRIPTION]
The combination of the scanning electron microscopy (SEM) with a focused ion beam (FIB), represents a pioneering and interesting tool to allow the investigation of the relationship occurring at the interface between cells and biomaterials. Herein, we provide a suitable protocol for cell-biomaterial sample preparation before imaging by SEM-FIB.

[STEPS]
SECTION: FIXATION
1. Remove culturing medium from the samples.
SECTION: FIXATION
2. Gently wash the samples twice in Phosphate Buffer Saline.
SECTION: FIXATION
3. Cover the surface of the samples with Gluthraldehyde 2.5% in Na-Cacodylate 0.1M buffer.
SECTION: FIXATION
4. Remove the Glutaraldehyde 2.5% in Na-cacodylate 0.1M buffer from the samples and cover the surface of the samples with Na-Cacodylate 0.1M buffer.
SECTION: FIXATION
5. Remove the Na-cacodylate 0.1M buffer from the samples.
SECTION: DEHYDRATION
6. De-hydrate the samples in Ethanol at increasing concentrations (35%, 55%, 70%, 90%, 95%). Each alcohol stands for 10 minutes at room temperature.
SECTION: DEHYDRATION
7. Store the samples in EtOH 95% at 4°C until further processing.
SECTION: DEHYDRATION
8. Cover the samples with Ethanol 99%.
SECTION: SPUTTER COATING
10. Sputter coat the samples for 30 seconds with Gold using a coating device 
SECTION: DEHYDRATION
9. Remove The Ethanol 99% from the samples and freeze dry the samples with Liquid Carbon Dioxide.
SECTION: SAMPLE OBSERVATION
11. Perform SEM-FIB analysis of the samples. We suggest to perform the SEM analysis at 5keV and to cross-section the samples with a Gallium ion beam accelerated at 30kV.
TITLE: lmmunostaining of Organoid Sections
AUTHORS: George Chen, Daniel H. Geschwind

[DESCRIPTION]
lmmunostaining of Organoid Sections derived from human iPS cells. 

[STEPS]
1. Place organoid sections into 1ml of PBS in a 24-well plate (1 section per well) 
2. Aspirate off the PBS gently
3. Add ~600ul of blocking buffer for 20mins at RT on the rocker
4. Wash 3x with 500ul of PBS and aspirate off PBS
5. Add primary AB at working concentration and incubate in Blocking Buffer overnight at 4°C (GFAP-1:500)
6. Wash 3x with 500ul of PBST, and aspirate it off
7. Add secondary AB at working concentration and incubate in Blocking Buffer at RT on a rocker for 1 hour, making sure to cover the plate with aluminum foil to prevent bleaching of the secondary AB (1:1000 for both secondaries)
8. Wash 3x in PBST mount onto a slide in water
9. DAPI staining is 2ug/ml in PBST for 15 min at RT 
11. Once the section is on the slide, aspirate off some of the water and add a drop of Fluoromount with DAPI before placing round coverslip on top, being careful not to cause any bubbles underneath
10. Wash 3x with PBST
12. leave overnight at RT to let dry and covered to prevent bleaching
13. Store covered at 4C
TITLE: No Title
AUTHORS: 

[STEPS]
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