eamag/protocols-with-steps huggingface dataset:
TITLE: GCaMP6F Fluorescence in Ex Vivo Intestinal Preparation
AUTHORS: Bryan  Yoo, Jessica  Griffiths, Sarkis  Mazmanian

[DESCRIPTION]
Protocol for GCaMP6F imaging in ex vivo intestinal tissue used in Yoo et al 2021

[STEPS]
1. 
Small intestinal tissue was quickly harvested from ChAT-Cre mice, flushed and placed in oxygenated (95% O2, 5% CO2), ice cold Krebs-Henseleit solution for 1 hour followed by 15 min at room temperature. 
2. A segment was cut along the mesenteric attachment, and pinned flat (mucosa facing down) on a Sylgard-lined recording chamber (Warner Instruments, PH1) in oxygenated Krebs-Henseleit solution.
3. C21 was added at 10nM and GCaMP6F fluorescence was detected on an upright microscope (Zeiss, Oberkochen, Germany-Examiner D1).
TITLE: Biochemical Measures of Neuropathy - Hydrogen Peroxide Assay
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">Oxidative stress is highly correlated with the metabolic changes caused by hyperglycemia. Increased levels of glucose overload mitochondria and result in the production of reactive oxygen species (ROS). In addition, the flow of excess glucose through cellular pathways decreases the cell’s normal ability to detoxify ROS. As a result, the neurons and axons of the peripheral nervous system contain increased levels of ROS and decreased antioxidant capacity. The following assays are used to measure these changes in rodent models of diabetic neuropathy. </div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/biy54kcn.jpg" /></div></div>

[STEPS]
?. [Sample Preparation:]
Prepare stock solutions above for Amplex Red Reagent, Reaction Buffer, HRP, 20 mM H²O² and Resorufin, sodium salt.
?. Prepare H²O² standard curve. Label tubes 1-5. Add 999 µL 1X Reaction Buffer to tube 2. Add 100 µL 1X Reaction Buffer to tubes 2 – 5. Add 1 µL of 20 mM H²O² working dilution prepared above to tube # 1 to produce concentrations of 20 µM. Take 100 µL from tube 1 and add to tube 2. Take 100 µl from tube 2 and add to tube 3. Take 100 µl from tube 3 and add to tube 4. Tube 5 is dH²O only. (Final concentration will be twofold lower, 0 to 10 µM.)
?. Dilute samples in 1X Reaction Buffer.
?. Pipette 100 µL of diluted standards, controls (if any) and samples into wells. (For DRG we used 25 µL)
?. From 20 µM stock solution of Amplex Red reagent - Prepare 400 µM dilution containing 2 U/mL HRP by adding 20 0µl of Amplex Red stock solution and 100 µL of 200 U/mL HRP stock solution to 9.7 mL of 1X Reaction Buffer.
?. Initiate reaction by adding 100 µL from above to each well.
?. Place plate into Fluroskan holder and click START.
?. Take 4 readings at 15 minute intervals using 544/590 filter pairs.
?. Save raw data as an Excel file into the RHPx data folder. Use the naming convention RHXXXX.xls, where XXXX is the date in mmdd format.
?. Select Process>Organize. Choose the appropriate data to organize (usually Measure1), then click OK. This rearranges the data into columns.
?. Save organized data as an Excel file into the RHP data folder. Use the naming convention rhXXXXor.xls, where XXXX is the date in mmdd format.
TITLE: 0.1M Phosphate Buffer
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to prepare 0.1M Phosphate Buffer. 0.1M Phosphate Buffer is used in the preparation of Sucrose solutions for cryoprotection.</div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>: Research reported in this publication was supported by the National Institute Of Mental Health of the National Institutes of Health under Award Number U19MH114830. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.</span></div></div>

[STEPS]
TITLE: Self Pollinating Tobacco
AUTHORS: Lynn Doran

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for self pollination of tobacco.  Validated on Petite Havana and Samsun varieties.</div></div>

[STEPS]
?. Grow tobacco plants to flowering.
?. Trim off lower florets of the chose inflorescence to create a clean section of stalk to attach the pollination bag to.
?. Choose an inflorescence to self pollinate.  Tobacco tillers and will generate multiple inflorescences per plant.  Any inflorescence will produce seed but the primary inflorescence will generate the most and the highest quality seed.
?. Remove any flowers that have started to open by pinching them off at their base. If you are unsure if a flower has opened too far or not, remove it.
?. A inflorescence ready for a pollination bag will have a long, clean section of stalk to attach the bag to and will have all opening flowers removed prior to bagging.
?. Place a pollination bag over the inflorescence.
?. Gently gather the bottom edges of the pollination bag just below the lowest florets, leaving as much space in the bag above the florets as possible.
?. Secure the bag with  a twist tie.  Do not overtighten as it may damage the plant.
?. Continue to care for the plant for several weeks to allow it to continue to produce pollen and self-pollinate the bagged flowers.
?. If required, tie the inflorescence to a stake in the pot to provide additional physical support as more flowers develop and the weight of the inflorescence increases.
?. When flowers fall off and the flower pods begin to brown, seeds are ready for harvesting.
?. Pollinating bags may need to be readjusted up the stalk as the plant grows to make room for additional flowers.
TITLE: Chimeric Protein-LAG and Streptococcal protein G sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between protein-LAG (PLAG) and streptococcal protein-G (SpG) with different immunoglobulin preparations of mammalian and avian species. </div></div>

[STEPS]
?. This ELISA was used to study the interactions between protein-LAG (PLAG) and streptococcal protein-G (SpG) with different immunoglobulin preparations of mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of PLAG in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of o-phenylenediamine solution (4 mg/mL) was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was set to 0.30.
TITLE: CD8 Cell Density In Substantia Nigra And Cerebral Peduncle Image Analysis
AUTHORS: Hemanth Ramesh Nelvagal, Toby J Curless, Zane Jaunmuktane

[DESCRIPTION]
QuPath is a bioimage analysis software designed for digital pathology and whole slide image analysis. This protocol describes how to measure CD8 density in the substantia nigra and cerebral peduncle using haematoxylin and DAB-stained brain sections.




[STEPS]
SECTION: Annotation
1. Manually annotate the substantia nigra and cerebral peduncle on NZConnect (Hamamatsu), a web-based whole-slide image (WSI) viewer. 
SECTION: Annotation
2. Download the annotations using a Python script then import into QuPath [1] using a Groovy script. 
SECTION: QuPath Deconvolution and CD8 Density Measurement
3. In QuPath, set the colour deconvolution to facilitate the detection of haematoxylin and DAB staining on CD3 IHC-stained sections.
SECTION: QuPath Deconvolution and CD8 Density Measurement
4. Segment all cell nuclei using StarDist [2] via the QuPath StarDist extension[3], follow with an object classifier to classify CD8-positive cells.

SECTION: QuPath Deconvolution and CD8 Density Measurement
5. Calculate CD8-positive cell density was calculated by the number of CD8-positive cells divided by the area of the region of interest (CD8-positive cells per mm^2).
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<strong>Supplemental notes:</strong><br /><br />An A<sub>260</sub> may be determined on a UV spectrophotometer (usually a 1:100 dilution works well).<br />1 A<sub>260</sub> unit of PBCV-1 routinely yields 1.5-2.5 X 10<sup>10</sup> PFU/ml of virus.<br /><br />For critical work, a second purification through sucrose gradients or a set of iodixanol gradients may be necessary.

[STEPS]
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TITLE: Protein Transfer
AUTHORS: Steven Burgess

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is based on the assumption that TGX pre-cast gels have been used, the procedure is very similar for self-made gels, just that the running time is longer. Using the transfer packs is economically similar to wet-transfer using MeOH.  </div><div class = "text-block"><span style = "font-weight:bold;">Note</span><span>:  Clean the apparatus immediately after use with dH</span><span style = "vertical-align:sub;">2</span><span>O and dry. This prevents the build-up of salts and rust which impair performance.  </span></div><div class = "text-block"><span style = "font-weight:bold;">Literature</span><span>: </span></div><div class = "text-block"><span>Full manual: </span><a href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10020688.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10020688.pdf</span></a><span style = ":;"> </span></div><div class = "text-block"><span>Quick-start: </span><a href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10016505D.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10016505D.pdf</span></a><span style = ":;"> </span></div><div class = "text-block"><span>Transfer pack: </span><a href="https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10019593D.pdf" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.bio-rad.com/webroot/web/pdf/lsr/literature/10019593D.pdf</span></a></div></div>

[STEPS]
?. Place the membrane and bottom stack in the middle of the cassette base
?. Place pre-cast gel in the middle of the membrane and roll to remove air-bubbles
?. Place the top stack on top of the gel, gently roll
?. Close the cassette lid, taking care not to disturb the gel
?. Place the cassette in the Trans-Blot Turbo and follow the instructions on the machine (Fast protocol TGX gel).
?. Either dry membrane and store at  for later use or proceed immediately to fluorescent western protocol
4 °C
TITLE: Germination and seedling establishment for deep-flow hydroponics: The benefit of slant boards
AUTHORS: noah.langenfeld, Bruce Bugbee

[DESCRIPTION]
Germination and seedling establishment for transplanting into hydroponics often uses porous substrates, but fine roots grow into these substrates, and they cannot be removed without damaging roots. Seedlings transplanted without removal of substrates can cause interactions with solution chemistry or addition of particulates to the nutrient solution. Germination of seeds on slant boards is clean, uniform, and reduces the time to transplanting. Slant boards facilitate long roots, which maximize exposure of the primary root to the nutrient solution after transplanting. The “boards” are made from thin acrylic or polycarbonate sheets with germination paper on top. Seeds are held in place by covering with thin paper before vertical placement of the boards in a container. Four to twelve days later, the seedlings with long roots can be removed from the paper without damage and transplanted into the hydroponic system. Here we describe slant board construction and procedures for rapid germination and transplanting.

[STEPS]
SECTION: Seeding
1. Clean a rigid plastic slant board and lay flat on a clean surface.
SECTION: Seeding
2. Cut a piece of germination paper 10 cm in height and the same length as the slant board.
SECTION: Seeding
3. Place germination paper on top of slant board.
SECTION: Seeding
4. Place seeds on top of germination paper (about 9 cm from the bottom of the board and 1 cm from the top of the germination paper) in a line parallel to the top and bottom of the slant board. Seeds should be about 5 mm apart.

SECTION: Seeding
5. Place a single layer of thin absorbent paper (Kimwipe) over the seeds.
SECTION: Seeding
6. Use a squirt bottle to gently wet the entire germination board and thin absorbent paper with water to lock seeds and paper in place on the board. Do not use nutrient solution.
SECTION: Seeding
7. Transfer the slant board into the exterior container. The angle of the board should be between 70° and 80°.
SECTION: Seeding
8. Fill the exterior container with purified water with calcium until the water level is about 2 cm above the bottom of the germination paper.
SECTION: Monitoring and transplanting
9. After at least one cotyledon leaf has appeared, gently peel back the thin absorbent paper to expose the leaves. The thin absorbent paper should remain covering the roots.
SECTION: Monitoring and transplanting
10. After the roots have grown long enough to reach the solution, remove the seedlings from the paper. Fingers or a tweezers can be used to remove the seedlings. Avoid excessive pressure to prevent root damage.
SECTION: Monitoring and transplanting
11. Place the seedling in a neoprene collar. Ensure the roots are not pinched in the collar. The cotyledons can be used to support the seedling and stop it from falling into the solution.
SECTION: Monitoring and transplanting
12. Place the neoprene collar with the seedling into the main culture tank. Ensure to roots can reach the nutrient solution to avoid desiccation.
TITLE: pIgNAT vector
AUTHORS: Glen Wheeler, Andrea Highfield, Rowena Stern

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">pIgNAT vector</div><div class = "text-block"><span>contains nourseothricin-resistance gene (NAT) codon-optimised for expression in </span><span style = "font-style:italic;">Isochrysis galbana</span><span>, flanked by promoter/terminator from </span><span style = "font-style:italic;">E. huxleyi </span><span> HSP70.</span></div></div>

[STEPS]
?.
TITLE: No Title
AUTHORS: 

[STEPS]
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TITLE: Fluorescent mutants screening in 96 well plates - Chlamydomonas reinhardtii
AUTHORS: Joao Vitor Molino

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is intended for screening experiments with algae cells. Nevertheless, with modifications, it can be used for other microrganims. </div><div class = "text-block">It is possible to use this protocol with deep-well plates, with modifications. </div></div>

[STEPS]
?. [Plate preparation]
For 96 well plates, the usual max volume is ~ . Clean and desinfect a biological cabinetPlace all materials inside. (e.g: sterile tips, media, sterile 96 well plates with lids, pipettes)Add   per well (Or another media) on the choosen plate. (For fluorescent experiments, black plates are recommeded. Clear bottom allows to simultaneously check absorbance)
330 µl
[TAP media]
?. [Colony picking]
Use sterile tooth picks to collect individual colonies. Place tooth pick into the wellProceed to the next colonyAfter collecting the desired number of colonies, mix the tooth pick in the media by spining itRemove and discard the used tooth picksVisually inspect the presence of green material inside the wells.
?. [Plate wraping ]
Add the lid to the plateWrapp it with a porous tape (Microporous), 3 laps around the plate, taping together the lid and plate.
?. [Growth]
Set the shaker to continuous mode, Illuminate with 60-80 µmols de photons/m2s, at Grow the cells for  (5 days) (Important to let cells grow enough to make the reads, 5-7 days have been tested)
Centrifuge: 900 33
25 °C
?. [Reading ]
Centrifuge the plates  to remove any condensation to the lid Add the plates to the microplate shakerSet the shaker to  , 900 RPMPlace plates in the Plate reader with the desired reading settings.Analyse the resultsSet the shaker to , 900 RPMPlace plates in the Plate reader with the desired reading settings.Analyse the results
Centrifuge: 2000 34, 3 min, 25 10
TITLE: Myco-Ed Module: Using iNaturalist - Create a Course Project
AUTHORS: Andrew W. Wilson

[DESCRIPTION]
This protocol is for instructors. Follow the steps for making a Traditional Project. This type of project will make it easier for instructors to control the observations that are included in the project and insure these are relevant to the course and Myco-Ed objectives.

[STEPS]
SECTION: Create a Traditional Project
1. We want to use a Traditional Project as opposed to a Collections Project on iNaturalist because the Traditional Project allows you and the students to have more control over which observations are included.

HOWEVER, iNaturalist requires that users have a minimum of 50 verifiable observations before they can create this kind of project. If you do not meet this requirement, please contact Andy (andrew.wilson@botanicgardens.org).

Click HERE to create a Traditional Project on iNaturalist.

  

SECTION: Create a Traditional Project
2. On the New Project Page start by...


 

SECTION: Create a Traditional Project
2.1. Create a New Project Title.
SECTION: Create a Traditional Project
2.2. Make the project invite-only.
SECTION: Create a Traditional Project
2.3. Add a project icon for your course.
SECTION: Create a Traditional Project
3. Scroll down to Observation Fields. 

Type "DNA Barcode ITS". When this option appears as an option in the menu, select it. It is then automatically added to project observations.
SECTION: Create a Traditional Project
3.1. Proceed to add other Observation Field as you see necessary. Adding Observation Fields will provide space for students to add data to each observation they include in the project. In the above case 

You can ignore the following sections:
Location - You can set this, but it's easier to have participants regulate which observations are added to the project.
Observation Rules - Same issue as with Location.
Project List - Not necessary.


SECTION: Create a Traditional Project
4. Select Create button at the bottom of the page.
SECTION: Create a Traditional Project
5. Your project is created!

Email Andrew Wilson (andrew.wilson@botanicgardens.org) upon completion of this step so it can be added to the Myco-Ed Umbrella Project on iNaturalist. You should also to the Myco-Ed Project on iNaturalist to become a member.

You are currently the only member of your course project. Go to the "Adding Students" Module for instructions on how to add students.
TITLE: Renal Multicompartment Segmentation Object Boundaries
AUTHORS: Nicholas Lucarelli

[DESCRIPTION]
Boundary descriptions for segmentations produced by the CMILab multicompartment segmentation pipeline for PAS-stained renal whole slide images. 

[STEPS]
SECTION: segmentations.ome.tiff channels
1. Cortical Interstitium: Defined as the spaces in the renal cortex situated between basement membranes of glomeruli, tubules, and vessels. Does not include perivascular stroma.
SECTION: segmentations.ome.tiff channels
2. Medulla Interstitium: Defined as the spaces in the renal medulla situated between basement membranes of tubules, and vessels. Does not include perivascular stroma.
SECTION: segmentations.ome.tiff channels
3. Non Globally Sclerotic Glomeruli: Outer boundary is defined by the outer boundary of the bowman's capsule. Glomeruli may show signs of sclerosis but are not deemed globally sclerotic.
SECTION: segmentations.ome.tiff channels
4. Globally Sclerotic Glomeruli: Outer boundary is defined by the outer boundary of what would be the bowman's capsule in a healthy glomerulus. Glomeruli are deemed globally sclerotic, and no longer functional.
SECTION: segmentations.ome.tiff channels
5. Tubules: Outer boundary is defined as the outer boundary of the tubular basement membrane. Classification is not specific to tubular segments (i.e. PCT, DCT, TAL, etc.).
SECTION: segmentations.ome.tiff channels
6. Arteries/Arterioles: Outer boundary is defined as the outer boundary of the medial layer of all arteries or arterioles in the sections. Veins and capillaries are not considered.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This study examined the effects of the ActiGraph’s (AG) low-frequency extension (LFE) filter on steps and physical activity classification in the free-living environment.  Thirty-four African-American women (age, 24.5±5.2 years; BMI, 24.9±4.5 kg/m<sup>2</sup>) had daily activity measured simultaneously with an AG-GT3X<sup>+</sup> accelerometer and a New Lifestyles NL-800 pedometer for seven days.  Steps per day (steps/d) and time (minutes/day) spent in sedentary, light, and moderate-to-vigorous physical activity (MVPA) were examined with and without the LFE filter (AG-LFE and AG-N, respectively). The AG-LFE recorded more total steps (13,723±4,983 steps/d) compared to AG-N and NL-800 (6,172±2,838 and 5,817±3,037 steps/d, respectively; <em>p</em>&lt;0.001).  Compared to the AG-N, the AG-LFE estimated less time in sedentary behaviors (518.7±92.1 vs. 504.2±105.4 min/d, respectively; <em>p</em>&lt;0.001), and more time in light (247.7±70.4 vs. 279.1±74.7 min/d, respectively; <em>p</em>&lt;0.001) and MVPA (18.9±16.9 vs. 21.5±18.2 min/d, respectively; <em>p</em>&lt;0.001), respectively. These data suggest that using the AG-LFE will affect steps and physical activity classifications. Future research should investigate the accuracy of these measures using the LFE filter.</p>

[GUIDELINES]
<p>Participants were enrolled in a larger cross-sectional study examining the relationship between daily physical activity intensity, cardiorespiratory fitness, and metabolic risk. As such, participants were excluded for any physical illnesses (cardiovascular or pulmonary disease, uncontrolled hypertension, or diabetes), pregnancy, smoking in the past six-months, or orthopedic disability that would limit their ambulatory function.  In an effort to capture the most typical physical activity measurements for each individual, participants were not permitted to enroll in the study if they were currently participating in any type of exercise or weight loss program that was adopted less than three months prior to entry into the study.  For the present study, participants were also excluded due to monitor failure or not meeting the minimum wear-time criteria.</p>

[STEPS]
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TITLE: CRISPResso analysis
AUTHORS: Hanqin  Li, Yogendra Verma, Dirk Hockemeyer, Frank Soldner

[DESCRIPTION]
This protocol describes the process of using CRISPResso to analyze Next Generation Sequencing (NGS) data from CRISPR-targeted, genome-edited samples. 



[STEPS]
1. Install CRISPResso2 
2. In linux, use the following command:

CRISPResso --fastq_r1 R1_reads.fastq.gz –fastq_r2 R2_reads.fastq.gz --amplicon_seq amplicon_seqeunce_here -g sgRNA_sequence_here
3. Use CRISPRessoBatch command if having many samples
4. For more details, https://github.com/pinellolab/CRISPResso2