eamag/protocols-with-steps huggingface dataset:
TITLE: B-3 BLOOD STORAGE
AUTHORS: REDI-NET Consortium

[DESCRIPTION]
This protocol describes blood storage.

[GUIDELINES]
OBJECTIVE

To outline steps for properly storing blood samples and nucleic acid samples purified from these samples.

SUMMARY/SCOPE

The overarching aim of the REDI-NET is to develop a collaborative laboratory network between domestic and international partnering institutions to address disease surveillance needs in order to effectively detect, predict and contain potentially emergent zoonosis. This SOP provides guidance on storage of blood samples and their purified nucleic acid to preserve their integrity for downstream nucleic acid extraction and/or sequencing library preparation


MAINTENANCE OF EQUIPMENT

Decontaminate a PCR workstation by keeping the UV light on for 15 min.
 


[STEPS]
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
1. 
 
Precool 96-well microfuge tube racks (for liquid forms including whole blood, plasma, serum, buffy coat) or sample box (for FTA cards only) on ice.

SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
2. Using preprinted adhesive labels or permanent markers, label 1.5 ml microfuge tubes or FTA cards with unique sample ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
4. Once the rack is full or all blood samples have been completed, label the rack with a unique rack ID.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
5. Close the rack lid tightly, secure with clear Saran wrap and immediately transfer to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
6. Update the freezer inventory so samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
7. 
 
In the clean PCR workstation, carefully transfer the eluted total nucleic acid to a 96-well PCR microplate, make sure to keep samples in the exact same locations corresponding to the rack where the original blood samples were stored.
 

SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
8. Cover the 96-well PCR microplate with adhesive film to prevent spill over or contamination.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
9. Label the film with a unique plate ID.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
10. Immediately transfer the 96-well PCR microplate to -80 °C freezer.
SECTION: STORAGE PROCEDURE FOR TOTAL NUCLEIC ACID
11. Update the freezer inventory so samples can be tracked properly.
SECTION: STORAGE PROCEDURE FOR UNTREATED SAMPLE
3. Transfer blood sample (liquid forms) into the corresponding pre-labeled 1.5 mL microfuge tubes and put it onto the microfuge tube rack (on ice) sequentially; or Transfer FTA card into a ziplock pouch with desiccant.
TITLE: Nextera XT at 0.2X On the Mantis
AUTHORS: Allen Institute for Brain Science

[DESCRIPTION]
Protocol to generate Nextera libraries using 0.2x reagents in a 96-well PCR plate using the Formulatrix Mantis instrument.

Note: 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.

[STEPS]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes how to make TRIS buffer (TRIS-HCl) at a 1M concentration and a pH 8.0</p>

[GUIDELINES]
<p>This protocol describes how to achieve a TRIS buffer at a pH 8.0. If a lower pH is required, i.e. pH 7.4, then add more hydrochloric acid until desired pH is reached</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[BEFORE_START]
<ul>
<li>Make sure you are using fresh <em>E. coli</em> cells streaked for isolation on LB + antibiotics no more than 1 week from -80°C cryostock.</li>
</ul>

[STEPS]
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TITLE: Fluorescent Immunolabelling for endogenous mouse LRRK2
AUTHORS: Roberta Marongiu, andrew.west west

[DESCRIPTION]
This protocol is used to label endogenous LRRK2 in mouse.
It has been edited from West et al., 2014
Protocol optimized for fresh fixed brain tissue sectioned at 40 um.

[STEPS]
2. Wash 3 times for 10 min in TBS 

3. Sections were “quenched” in 100% Methanol for 15 min at 4 °C  on shaker.

4. Wash 3 times for 10 min in TBS

5. Incubate the slices with Sodium Citrate – 10 mM Sodium Citrate pH 6.0 w/0.05% Tween for 30 min  at 37 °C  on shaker.

6. Wash 3 times for 10 min in TBS
7. During the washes, prepare the blocking solution : 5% normal donkey serum (matched to secondary AB) in TBS with 0.3% Triton
8. Incubate the slides with blocking solution for 60 min  at 4 °C on shaker.

9. Wash 3 times for 10 min in TBS
10. During the washes, prepare the primary antibody solution : 5% normal donkey serum in TBS with NO TRITON + Primary Antibody anti-LRRK2 (1:500 or 1:1000)
11. Incubate at 4 °C on shaker for 1440 min 2880 min 

12. Wash 3 times for 10 min in TBS
13. During the washes, prepare the secondary antibody solution : 5% normal donkey serum in TBS with NO TRITON + Alexa Fluor secondary AB (1:1000)
14. Incubate at 4 °C on shaker for 1080 min 

15. Wash 2 times for 5 min in TBS
16. During the washes, prepare the DAPI solution :   DAPI (1mg/ml): 1:10,000, in TBS
17. Incubate the slides with the DAPI solution for 10 min 

18. Wash 3 times for 10 min in TBS
19. Mount sections on superfrost + slides and coverslip with Invitrogen Prolong Antifade reagent.
1. Select brain slices, from fresh, unfrozen, fixed tissue - slices are 40um thick
TITLE: Coverslip Functionalization SOP003.v2.2
AUTHORS: Rory  Kruithoff, Douglas  Shepherd

[DESCRIPTION]
Document Summary:
This document, SOP003 – Coverslip Functionalization, describes the process for cleaning and modifying the coverslip surface for improved adhesion of a sample.This procedure describes two methods of functionalization, silanization and poly-d-lysine (PDL) coating.  Silanization is a method used to promote covalent adhesion of polyacrylamide gel-embedded tissue while poly-d-lysine (PDL) coating of silanized coverslips provides a protein anchoring system which improves adhesion of living cells to the surface of the coverslip. 

Quick Overview:
Part 1 - Clean coverslips 
Part 2 - Silanize coverslips 
Part 3 - PDL coat coverslips 

[BEFORE_START]
Using 40-mm-diameter #1.5 coverslips

[GUIDELINES]
v2.2 revision notes
-Added Bottiger lab and OsmFISH references.


References:
Moffitt, J. R., Hao, J., Bambah-Mukku, D., Lu, T., Dulac, C., & Zhuang, X. (2016). High-performance multiplexed fluorescence in situ hybridization in culture and tissue with matrix imprinting and clearing. Proceedings of the National Academy of Sciences, 113(50), 14456-14461. https://doi.org/10.1073/pnas.1617699113

Boettiger Lab Github: https://github.com/BoettigerLab/protocols/blob/master/MatrixEmbedding.md

OsmFISH dx.doi.org/10.17504/protocols.io.psednbe

[STEPS]
SECTION: Part 1 - Clean coverslips
1. Wash for 30 min via immersion in 1:1 mix of 37 % (v/v) and methanol at Room temperature.
SECTION: Part 1 - Clean coverslips
2. Rinse 3x in DI H2O.
SECTION: Part 1 - Clean coverslips
3. Fill beaker with Rnase-free water and autoclave coverslips to sterilize.
SECTION: Part 1 - Clean coverslips
4. Rinse 1x in 70 %.
SECTION: Part 1 - Clean coverslips
5. Coverslips can be stored in 70 % indefinitely.
SECTION: Part 2 - Silanize coverslips
6. Dry at 60 °C.
SECTION: Part 2 - Silanize coverslips
7. Immerse in silanization solution in Fume-hood for 30 min at 60 Room temperature.
SECTION: Part 2 - Silanize coverslips
8. Wash 1x with chloroform.
SECTION: Part 2 - Silanize coverslips
9. Wash 1x with ethanol.
SECTION: Part 2 - Silanize coverslips
10. Bake in a 60 °C oven for 120 min to dehydrate the silane layer.
SECTION: Part 2 - Silanize coverslips
11. Remove from oven and let coverslips cool.
SECTION: Part 2 - Silanize coverslips
12. Store in a desiccated chamber for weeks.
SECTION: Part 3 - PDL coat coverslips (for cell culture on coverslips)
13. Immerse coverslips in PDL Solution for 60 min at 60 Room temperature.
SECTION: Part 3 - PDL coat coverslips (for cell culture on coverslips)
14. Rinse coverslips three times with nuclease-free water and then dry at 60 Room temperature.
SECTION: Part 3 - PDL coat coverslips (for cell culture on coverslips)
15. Silanized, PDL-coated coverslips may be stored at 60 Room temperature in a desiccation chamber for weeks.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p><strong>The architecture of normal and diseased tissues strongly influences the development and progression of disease as well as responsiveness and resistance to therapy. We describe a tissue-based cyclic immunofluorescence (t-CyCIF) method for highly multiplexed immuno-fluorescence imaging of formalin-fixed, paraffin-embedded (FFPE) specimens mounted on glass slides, the most widely used specimens for histopathological diagnosis of cancer and other diseases.  t-CyCIF generates up to 60-plex</strong> <strong>images using an iterative process (a cycle) in which conventional low-plex fluorescence images are repeatedly collected from the same sample and then assembled into a high dimensional representation. t-CyCIF requires no specialized instruments or reagents and is compatible with super-resolution imaging; we demonstrate its application to quantifying signal transduction cascades, tumor antigens and immune markers in diverse tissues and tumors. The simplicity and adaptability of t-CyCIF makes it an effective method for pre-clinical and clinical research and a natural complement to single-cell genomics. </strong></p>

[GUIDELINES]
<p>Key resources, reagents and software used in this study are listed on-line at the HMS LINCS Center Publication Page <a href="http://lincs.hms.harvard.edu/lin-elife-2018/" target="_blank" rel="noopener noreferrer">http://lincs.hms.harvard.edu/lin-elife-2018/</a> (RRID:SCR_016370).  This page provides links to an OMERO image database from which individual images can be obtained; stitched and registered image panels can be obtained at <a href="http://www.cycif.org" target="_blank" rel="noopener noreferrer">www.cycif.org</a>.(RRID:SCR_016267) and a video illustrating the t-CyCIF method can be found at <a href="https://vimeo.com/269885646" target="_blank" rel="noopener noreferrer">https://vimeo.com/269885646</a>.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is based on Schleper et al. (1992) as modified by Stedman et al. (2003).<br /><br />This is a protocol from: <br /><br />Stedman, K. M., K. Porter, and M. L. Dyall-Smith. 2010. Chapter 6: The isolation of viruses infecting Archaea. Manual of Aquatic Viral Ecology. Waco, TX:American Society of Limnology and Oceanography. doi:10.4319/mave.2010.978-0-9845591-0-7<br /><br />Please see the <a href="http://www.aslo.org/books/mave/MAVE_057.pdf" target="_blank">published manuscript</a> for additional information.

[GUIDELINES]
<strong>Authors: </strong>Kenneth M. Stedman<sup>1</sup>, Kate Porter<sup>2</sup>, and Mike L. Dyall-Smith<sup>3</sup> <br /><sup>1</sup>Department of Biology, Center for Life in Extreme Environments, Portland State University, P.O. Box 751, Portland, OR 97207, USA <br /><sup>2</sup>Biota Holdings Limited, 10/585 Blackburn Road, Notting Hill Victoria 3168, Australia <br /><sup>3</sup>Max Planck Institute of Biochemistry, Department of Membrane Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany<br /><br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/bwrc2xe.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/bwqc2xe.png" data-ofn="MAVE ch 6 fig 1.png" /><br /><strong>Fig. 1:</strong> Pictorial overview of isolation of <em>Sulfolobus</em> viruses. <br />(A) Wolfram Zillig sampling at a typical <em>Sulfolobus</em>-containing pool in Yellowstone National Park, USA, September 2000 (inset shows anaerobic tubes with samples). <br />(B) 80°C incubator with long-necked growth flasks (detail in inset). <br />(C) Singlecolony isolates of <em>Sulfolobus solfataricus</em> on a Gelrite® plate. This plate contains a mixture of <em>S. solfataricus</em> containing (blue colonies) and lacking (brown) a vector expressing the <em>lacS</em> gene from <em>S. solfataricus</em> and was sprayed with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) (see Jonuscheit et al. 2003). <br />(D) Lawn of <em>S.solfataricus</em> strain P1 with halos of growth inhibition due to virus production by 2-µl spots of virus-infected strains. Spots labeled SV2P1 and SV2P2 are from <em>S. solfataricus</em> strains P1 and P2 infected with SSV-I2 respectively (Stedman et al. 2003). Spot labeled C is a detergent-positive control. Spot labeled P2- is uninfected <em>S. solfataricus</em> strain P2 as a negative control.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Biodiversity data exist in large quantities, which is a boon to biodiversity science. In spite of large numbers of data records being available, however, the proportion of those records that is readily usable for science applications is quite small. The difference between the full number of data records existing <em>versus </em>the records that are ready for use is the result of what we call "leakage" of data, in the form of steps that have not been taken or errors that remove the utility of the data. In this contribution, we explore several large-scale biodiversity data sets in terms of why they contain data records that are or are not ready for use.</p>

[GUIDELINES]
<p><strong>GBIF Data Precautions</strong>: We note that data from the GBIF data portal are subjected to the GBIF taxonomic filtering (Gaiji et al. 2013), although our experience indicates that the GBIF filters apply to species-based searches, but not to database-level or region-based searches, such that the data analyzed herein have not to our knowledge been subjected to these filters. For the Brazilian Virtual Herbarium, names are from Brazilian Flora 2020 (http://floradobrasil.jbrj.gov.br) and Catalogue of Life (http://www.catalogueoflife.org/), in that order). We did not consider the potential for an expert to review and identify the specimen fully as rescuable, as that step would extend beyond the data to actual handling of the specimen, or at least detailed inspection of images by specialists.</p>
<p> </p>
<p><strong>Georeferencing Caveats</strong>: We required that georeferences demonstrate the characteristics of best practice, as outlined in the MaNIS georeferencing protocol. These data were considered as complete only when geographic coordinates were accompanied by full metadata, such that information was present in the fields coordinatePrecision and coordinateUncertaintyInMeters, as this information is crucial to many applications of these data in biodiversity informatics applications, preventing misinterpretation of coarse-resolution coordinates.</p>

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Separates molecules based on size. Great for checking DNA after a Restriction Digest. This protocol is for a 1.2% agarose gel, sufficient for resolving DNA 400bp to 7kb (conservatively). The gel electrophoresis system is a 7.5cm x 10cm gel bed area with approximately 15cm electrode distance. The minimum volume required for a 7.5cm x 10cm x 0.5cm gel is 37.5mL. The maximum gel height for 50mL is 0.67cm.

[BEFORE_START]
Have a DNA Sample ready, typically either from PCR or a recently performed Restriction Digest. Dilute down the 50X TAE Buffer to 1X using sterile filtered water. For the dual-sided 1.0mm gel comb MHC-10-0816 the maximum volume is 35/13μL. For the dual-sided 1.5mm gel comb MHC-15-1014, the maximum sample volume is 38/25μL.

[STEPS]
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TITLE: Immunohistochemical labeling of thick cryosections from pelvic ganglia
AUTHORS: Janet Keast, Peregrine Osborne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes immunohistochemical procedures applied to thick (50 µm) cryosections mounted directly on slides. It is used when the structures to be analysed are too large to remain intact within thin (10-20 µm) cryosections. Antibodies have been selected to distinguish different neurochemical classes of autonomic ganglion neurons and synaptic boutons associated with these neurons. The protocol can also be used to characterize neurons containing retrograde tracer.</div></div>

[STEPS]
?. [Preparation of cryosections]
Cryoprotect fixed tissue in phosphate-buffered saline (PBS; 0.1 M, pH7.2) containing 30% sucrose. This should be performed at 4C, 24-72h prior to cutting.
?. [Preparation of cryosections]
Embed tissue in cryomold using OCT, freeze in cryostat and cut sections (50 µm), distributing sections progressively across sets of 3-4 slides so that each slide has non-consecutive sections.
Chrome-alum treated slides are pre-coated (subbed) with 1% gelatin, dried and stored at room temperature. This slide treatment enhances section adhesion while retaining good surface tension of the antibody droplets.
?. [Immunostaining]
Air-dry slides at room temperature for at least 10 minutes.
?. [Immunostaining]
Wash sections in PBS (10 min).
?. [Immunostaining]
Incubate sections in blocking solution (PBS containing 10% non-immune horse serum and 0.5% triton X-100) at room temperature in a humidified dark chamber for 2 h.
?. [Immunostaining]
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 48h in a humidified dark chamber. Antibodies are diluted in PBS containing 2% horse serum, 0.5% Triton and 0.1% sodium azide.
?. [Immunostaining]
Wash sections in PBS (30 min)
?. [Immunostaining]
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) for 4h in a humidified dark chamber. Antibodies are diluted in PBS containing 2% horse serum, 0.5% Triton and 0.1% sodium azide.
?. [Immunostaining]
Wash sections in PBS (30 min)
?. [Immunostaining]
Coverslip in Vectorshield or preferred anti-fade mountant.
?. [Immunostaining]
Draw around sections with hydrophobic barrier pen (PAP pen); wait for 10 min to dry.
TITLE: Selection of Stationary Phase of HPLC for Posaconazole Estimation
AUTHORS: annamalai.rama

[DESCRIPTION]
Introduction: Posaconazole is a widely used antifungal drug, and its accurate quantification is essential for quality control and assessment of its pharmaceutical products. This study aimed to develop and validate a reverse-phase high-performance liquid chromatography (HPLC) analytical method for quantifying Posaconazole in bulk and dosage form.

Methods: The HPLC method was developed and validated based on International Conference on Harmonisation (ICH) guidelines. The developed method was then applied to quantify Posaconazole in a marketed tablet formulation. The method's specificity, linearity, precision, accuracy, robustness, and stability were evaluated.

Results: The developed HPLC method showed good linearity over a 2-20 μg/mL concentration range. The percentage recovery of Posaconazole from the bulk and marketed formulations was found to be 99.01% and 99.05%, respectively. The intra-day and inter-day precisions were less than 1%, and the method was stable under different conditions. The HPLC method was successfully applied to quantify Posaconazole in the marketed formulation.

Conclusion: The developed and validated HPLC method is reliable and efficient for analyzing Posaconazole in bulk and dosage forms. The method's accuracy, precision, specificity, linearity, robustness, and stability demonstrate its effectiveness. The method can be used for the quality control and assessment of Posaconazole-containing pharmaceutical products.

[STEPS]
SECTION: Selection of Stationary Phase
1. To determine the appropriate stationary phase for the HPLC method, a total of 60 trials were conducted using various columns, including Phenomenex Luna C18, Phenomenex Kinetex C18, Phenomenex Hyperclone C18, and Agilent Eclipse XDB C18.
SECTION: Selection of Stationary Phase
2. The selection criteria included cost-effectiveness, sensitivity, retention time, peak area, tailing factor, and K factor.
SECTION: Selection of Stationary Phase
3. After a thorough evaluation, the Phenomenex Hyperclone C18 column was chosen as the optimal stationary phase due to its superior performance and cost-effectiveness.
SECTION: Selection of Stationary Phase
4. The results of this selection process ensured that the HPLC method was optimized to deliver accurate and reliable results for the quantification of Posaconazole in bulk and dosage form.
TITLE: “Leveraging AI Tools to Bridge the Healthcare Gap in Rural Areas in India&#34;
AUTHORS: Ajit Kerketta

[DESCRIPTION]
Introduction:
Despite considerable progress in the healthcare sector, rural areas continue to grapple with healthcare deficiencies, which eventually impact the quality of health outcomes. However, the emergence of AI technology offers promising solutions to overcome these hurdles. Hence, the study explores the potential and efficacy of introducing artificial intelligence (AI) tools to address the healthcare disparity in rural India.
Methods:
The research employed a literature review method and gathered data from various databases such as Science Direct, PubMed, and Google Scholar. The screening process was aided by the Rayyan electronic software. Articles published in
English between January 2020 and December 2022 were selected, followed by a thematic analysis of the findings.
Results:
Results indicate the potential of AI in rural healthcare settings, showing AI-driven solutions addressing healthcare access gaps and contributing to their bridging. The study also highlights hurdles related to AI tool adoption in rural healthcare and proposes collaborative efforts among policymakers, healthcare providers, and technology developers to integrate AI tools
effectively. This necessitates advocating for digital infrastructure investments, capacity-building initiatives, and conducive regulatory frameworks for AI implementation.
Conclusion: The study underscores AI's transformative role in bridging the healthcare gap in rural India. By harnessing AI technologies, healthcare providers and policymakers can surmount barriers, empower local healthcare workers, and improve health outcomes for rural communities The insights and recommendations contribute to the evolving knowledge base on leveraging AI for adequate healthcare delivery, guiding future initiatives in similar contexts.
 
Keywords:
Artificial Intelligence, Healthcare gap, Rural areas, remote patient monitoring, healthcare outcome.  

[STEPS]
SECTION: “Leveraging AI Tools to Bridge the Healthcare Gap in Rural Areas in India&quot;
1.
TITLE: Protocols for "Bicolor Angelfish (Centropyge bicolor) genome provided first chromosome-level reference of Pomacanthidae family and clues for bi-color body formation"
AUTHORS: Chunhua Li, Xianwei Yang, Libin Shao, Rui Zhang, QunLiu, Mengqi Zhang, Shanshan Liu, Shanshan Pan, Weizhen Xue, Congyan Wang, Chunyan Mao, He Zhang, Guangyi Fan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>The bicolor angelfish, </span><span style = "font-style:italic;">Centropyge bicolor,</span><span> is a kind of tropical coral reef fish that is also well-known as bicolor angelfish because of its special and beautiful two-color body. However, the mechanism of bicolor body has not been studied clearly due to the lack of high-quality genomic data. Here we assembled a chromosome-level genome of the bicolor angelfish with a total length of 650 Mb, contig N50 of 114 Kb and scaffold N50 of 4.4 Mb. Based on the genome, 21,774 protein coding genes were annotated. Furthermore, we identified several candidate gene families associated with its bicolored body developing. As we are known, our study is the first provides a chromosome-level genome as reference for the family Pomacanthidae, which would contribute to the future researches of colorful fish.</span></div></div>

[STEPS]