eamag/protocols-with-steps huggingface dataset:
TITLE: Hematoxylin &amp; Eosin Protocol For Leica ST5020 Automated Stainer
AUTHORS: Angela Denn

[DESCRIPTION]
This Hematoxylin and Eosin protocol is for the Leica ST5020 Automated multistainer. You can use it as a guide to developing your own protocol, catered to the needs of your researchers. This template can be modified for manual staining but best and most reproducible results are easier to achieve with an automated stainer. 

[GUIDELINES]
 Daily Maintenance for ST5020
 
 Filter reagent cup 21 (Hematoxylin 560) using a funnel, Whatman #1 filter paper, and 500ml Erlenmeyer flaskDispose of reagent cup 1 (Pro-par) into aqueous wasteMove reagent cup 2 into the 1 positionMove reagent cup 3 into the 2 positionFill a clean, dry reagent cup with fresh Pro-par and place in reagent cup 3 positionDispose of reagent cup 4 (100%IPA) into aqueous wasteMove reagent cup 5 into the 4 positionFill a clean, dry reagent cup with fresh (100%IPA) and place in reagent cup 5 positionDispose of reagent cup 6 into aqueous waste and replace with fresh 90%IPADispose of reagent cup 33 (Define) into aqueous waste and replace with fresh DefineDispose of reagent cup 32 (Blue Buffer 8) into aqueous waste and replace with fresh Blue Buffer 8 in a clean, dry reagent cupDispose of reagent cup 31 (70% IPA) into aqueous waste and replace with fresh 70% IPADispose of reagent cup 29 (90% IPA) into aqueous waste and replace with fresh 90% IPADispose of reagent cups 16 and 17 (100% IPA) into aqueous wasteMove reagent cup 15 into the 17 positionMove reagent cup 28 into the 16 positionMove reagent cup 27 into the 15 positionFill clean, dry reagent cups with fresh 100% IPA and place into the 27 and 28 positionAdd approximately 20 grams of DriRite to reagent cups 27 and 28Dispose of reagent cup 26 (50:50 Propar/100% IPA) into aqueous wasteFill clean, dry reagent cup with 50:50 Propar/100% IPA. Add ~20 grams of DriRite and place in position 26Dispose of reagent cup 14 (Pro-par) into aqueous wasteMove reagent cup 13 into the 14 position and add ~20 grams of Dri-RiteMove reagent cup 25 into the 13 positionFill clean, dry reagent cup with fresh Pro-par and place in position 25Record maintenance performed on the maintenance sheet
 Weekly Maintenance for ST5020
 
 Remove cups 7, 8, 9, 10, 11, and 12Hand wash in hot soapy water using a sponge and Dawn dish detergentRinse thoroughly in hot waterSoak cups in a 10% bleach solution for ten minutesRinse thoroughly in hot waterReplace cups into positionRecord maintenance performed onto the maintenance sheet
 Monthly Maintenance for ST5020
 
 Dispose of reagent cup 21 (Hematoxylin 560) into aqueous wasteFill clean, dry reagent cup with fresh Hematoxylin 560 and place into the 21 positionDispose of reagent cup 18 (Eosin Phloxine 515) into aqueous wasteFill clean, dry reagent cup with fresh Eosin Phloxine 515 and place into the 18 positionRecord maintenance performed on the maintenance sheet
 

 

[STEPS]
SECTION: H&amp;E Stain
1.  
  
Reagent layout for multi stainer use.
SECTION: H&amp;E Stain
2.   
Program for H&E protocol.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This protocol describes the generation of mFwe Knockout n2a cells. It is from 'Flower Ca<sup>2+</sup> channel in CME and ADBE' of Yao CK et al.</p>
<div>
<div>
<p> </p>
<p>Please see the manuscript for the full method details.</p>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: lysis buffer裂解基因组
AUTHORS: 张 雪

[STEPS]
?. 〔0.5µl Proteinase K＋20µl lysis buffer，1:40混合〕/embryo，55°C，5h
55 °C
?. 95°C，15min，此为stock
95 °C
?. 吸取3µl stock+47µl ddH2O，此为work
TITLE: Ambient sample storage system of field-collected insect samples for genomics
AUTHORS: Roland Wouters, Sam Mugford, Saskia Hogenhout

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Population genomics studies require the purification of high-quality DNA from field collected samples for subsequent analysis. Often it is impossible or impractical to snap freeze collected samples. Alternate storage methods have been used, such as preserving specimens in ethanol or DMSO. However, both are detrimental to the quality of the DNA recovered. We have found that dehydrating field-collected insect samples using silica gel is a simple, cheap and effective alternative. We can recover high quality DNA suitable for short-read sequencing approaches from samples that have been stored with silica gel for up to 8 weeks stored at ambient temperatures- suitable for the most remote field studies. </div><div class = "text-block">Here we present a method for the construction of low-cost sample collection and storage tubes from commonly available laboratory consumables.</div><div class = "text-block"><span class = "embed-element"><iframe width="560" height="315" src="https://www.youtube.com/embed/L2nlGcUvW14" frameborder="0" allow="accelerometer; autoplay; encrypted-media; gyroscope; picture-in-picture" allowfullscreen></iframe></span></div></div>

[STEPS]
?. Measure the pipette tip against the Eppendorf tube, and cut off the tip of the pipette tip. The remaining length of the pipette tip should fit inside the Eppendorf tube, with the wide end of the tube fitting tightly against the inside of the tube.
?. With the tip removed from the tube, add approximately 100ul of silica gel to the tube.
?. Push the tip into the tube through the silica gel. Close the tube.
?. When collecting samples, place the insect sample into the wide end of the tip, close the tube lid.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<div title="Page 53">
<div>
<div>
<p>Standard hematological analyses</p>
<p>EDTA-anticoagulated blood was used for cytometric analysis using a whole blood counter Sysmex XE 2100® (Sysmex,Kobe, Japan) to obtain a whole blood count. Fibrinogen levels were determined with an ACL-9000 (Diamond Diagnostics, Holliston, MA) coagulation analyser, using a PT-fibrinogen high sensitivity reagent, which is a high-sensitivity calcium thromboplastin that allows the simultaneous determination of PT and fibrinogen levels. aPTT was measured using an ACL-9000 coagulation analyser and INR was calculated by the formula INR (PT patient/PT normal).</p>
</div>
</div>
</div>

[STEPS]
TITLE: Sampling leaf tissue for analysis of NPQ Relaxation using  Technologica Chlorophyll Fluorescence Imager Data.
AUTHORS: Lynn Doran

[DESCRIPTION]
Sampling leaf tissue for analysis of NPQ Relaxation using  Technologica Chlorophyll Fluorescence Imager Data.

[STEPS]
SECTION: Leaf Tissue Sampling
1. Cut #1 Whatman filter paper into small squares that are just bigger than the well on a 24-well culture plate.
SECTION: Leaf Tissue Sampling
5. Using a #2 Humboldt cork borer, take leaf tissue samples from each plot for each technical replicate.  Hold the leaf flat against the rubber stopper, press the cork borer through the leaf into the stopper and twist.  Try to use the most recently fully expanded leaf with mature chloroplasts and limited bug damage.  Avoid sampling over the midrib.

If taking multiple technical replicates from a plot, multiple leaf discs can be sampled simultaneously and will continue to move up the cork borer.
 

SECTION: Leaf Tissue Sampling
4. Label the lid of the plate and the side of the plate with the plots sampled.  
 

SECTION: Leaf Tissue Sampling
6. Using a long cotton swab, or spatula, push the leaf discs out of the cork borer into the appropriate well of the 24 well plate.  All technical replicates can be in the same well but each plot (biological replicate) must go in it's own well.
SECTION: Leaf Tissue Sampling
7. When a complete 24 well plate has been sampled, place the lid on.  Store the plate out of direct sunlight at room temperature. 

Typically a bag, box, or empty cooler (no ice) works well.
SECTION: Leaf Tissue Sampling
8. When all plates have been collected, return to lab.
SECTION: Leaf Tissue Sampling
9. Using tweezers, transfer each leaf disc to an individual well in a 96 well plate.  The top  of the leaf disc should be facing down on to the bottom of the 96 well plate.

Handle the leaf discs gently.  Damage to the tissue, i.e. being poked with tweezers, will affect chlorophyll fluorescence analysis.
 

SECTION: Leaf Tissue Sampling
10. Dip a nasal aspirator filter halfway into water and then immediately dab on a paper towel.  Insert filter in the well on top of the leaf disc.  Gently press the nasal aspirator filter all the way to the bottom of the well, ensure that the leaf disc is flat on the bottom of the 96-well plate and that the damp filter is touching the leaf disc.

 

SECTION: Leaf Tissue Sampling
11. When all wells have leaf discs and sponges inserted, place the lid on the plate.  Tape the top right corner to help orient the plate in the dark for imaging.
SECTION: Leaf Tissue Sampling
12. Parafilm the lid to the plate.

SECTION: Leaf Tissue Sampling
13. Wrap the plate in aluminum foil and write the plot IDs and plate ID on the aluminum foil.
SECTION: Leaf Tissue Sampling
14. Place in a dark box or cabinet for a minimum of 1 hour, but preferably up to 12 hours before imaging and analysizing per Fluorescence analysis using CF imagerV.2.
SECTION: Leaf Tissue Sampling
2. Use the #7 cork borer to push two small squares of #1 Whatman filter paper into each well of the 24-well culture plate.
 
SECTION: Leaf Tissue Sampling
3. Thet the filter paper using tap water.
TITLE: Analogizing the Nexus of Nutritional Status and Oral Health in Children of Pre-School age in Ranchi City of East India: A Protocol
AUTHORS: Arunima, dr annapurna ahuja, drvipinahuja, nilima dr nilima thosar

[DESCRIPTION]
This contemporary research purposes to analogize the nexus between the Body mass index (BMI) and oral health status (caries, plaque status) in primary dentition of preschool going children of Ranchi, Jharkhand, India. No significant association was concluded between BMI, dental caries and plaque aggregation in pre-school children of Ranchi city. The interconnection of BMI with dental caries and plaque accumulation is always seen with two sided documented
literature, one section proves positive or negative association, second proves no association between these parameters. Our study adds to the previous literature proving no connotation between BMI, dental caries and plaque aggregation.


[STEPS]
SECTION: Introduction
1. 
Anthropometric measurements are the human body
measurements which quantify us with vital indicators of nutritional status in
children and adults. These include overall health, nutritive adequacy, growth
and development measurements over time in growing children and adults. Body
mass index (BMI) measurement index
is used for measuring nutritional status quantitatively by measuring height and
weight respectively and can be compared with other parameters like oral
diseases. Voluminous research had been conducted so far to compare BMI with dental caries and periodontal diseases. However,
there is a scarcity of literature in the arena of pediatric dentistry as far as
BMI relationship with dental caries or oral hygiene standing in pre-school
children is concerned. In fact, only handful of studies of such nature had
been attempted so far in Jharkhand region of India. Thus, this research
work is a genuine endeavor to recognise the association between nutritional
status and dental health in pre-school children in Jharkhand region.
TITLE: Biochemical Measures of Neuropathy - Lowry Protein 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/biy94kcn.jpg" /></div></div>

[STEPS]
?. [Performing the Assay:]
1. Thaw samples on ice. 2. If samples contain detergent: add 20ul of reagent S to each ml for reagent A 3.  Prepare standard as follows: 4. Pipet 5 µl of standards and samples into plate. 5. Add 25 µl of reagent A to each well. 6. Add 200 µl reagent B to each well. 7. Place plate in reader and press START.
?. [Reading the Plate – (wavelength 750 nm)]
1. Wipe the bottom of plate to remove finger prints, dirt, etc. 2. Open ascent software to saved template. Place plate onto Multiskan holder and click START. Plate will shake for 5 seconds then incubate 15 min. then read plate at 750 nm. 3.  Save raw data as an Excel file into the Lowry data folder. Use the naming convention LYXXXXXX.xls, where XXXXXX is the date in yymmdd format. 4.  Select Process>Organize. Choose the appropriate data to organize (usually Measure1), then click OK. This rearranges the data into columns. 5.  Save organized data as an Excel file into the LY data folder. Use the naming convention LYXXXXor.xls, where XXXX is the date in mmdd format.
TITLE: Adult mouse skin dissociation protocol (on ice)
AUTHORS: Andrew Potter

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol was developed to dissociate adult (8-10 wk) mouse skin "on ice". It utilizes two layers of digestion with a </span><span style = "font-style:italic;">Bacillus licheniformis </span><span>protease cocktail, combined with mechanical disruption from a dounce homogenizer. The cell yield is 3000 cells/mg. </span></div></div>

[STEPS]
?. [Isolating tissue]
After euthanizing mouse, remove hair using Nair: dab with Nair, wait 30 secs, wipe with wet paper towel.
?. [Isolating tissue]
Isolate tissue and place in ice-cold hypothermosol.
?. [Isolating tissue]
Scrape off underlying layer of fatty / connective tissue using scalpel before proceeding.
?. [Isolating tissue]
Mince skin tissue thoroughly on petri dish on ice for 3-4 min on ice into 1-mm3 pieces using razor blade while manipulating tissue with forceps – you will need to use grinding motion and vigorously break up tissue.
[mincing]
?. [1st digest layer]
Place 23 mg minced tissue into 1 mL B. Lich enzyme cocktail. Incubate on ice.
[minced tissue]
?. [1st digest layer]
Shake every min; triturate 10x every 2 min with p1000 w/tip cut (start triturating at 2 min) for 20 min.
[digest]
[shake]
[triturate 10X]
?. [1st digest layer]
After 20 mins of triturating on ice, use pipet to transfer digest mix to 2 mL dounce homogenizer. Use 10 strokes of Pestle A every 2 min (4 series total, 8 min). Digest mix should become turbid.
[dounce homogenize]
[digest using dounce]
?. [1st digest layer]
Transfer back to 1.5 mL tube using 1 mL serological pipet. Mix thoroughly and allow to settle on ice 2 min.
[settle on ice]
?. [1st digest layer]
Save 70% (700 µL) of supernatant, leaving chunks at the bottom of the tube; apply to 30 µM filter on 15 mL conical. Rinse filter w/5 mL ice-cold PBS/BSA 0.04%. Save flow through on ice and keep filter on tube for 2nd layer.
[save supernatant]
[rinse filter w/ice-cold PBS/BSA 0.04%]
?. [2nd digest layer]
Add additional 1 mL b. Lich enzyme mix to residual tissue chunks.
[b. lich enzyme mix]
?. [2nd digest layer]
Triturate 10x every 2 min, shake every min while incubating on ice for 20 additional mins. (50 min. total digest time).
[additional digest time]
[triturate 10x]
[shake every min.]
?. [2nd digest layer]
Transfer entire volume to same 30 µM filter on 15 mL conical. Rinse with additional 5 mL ice-cold PBS/BA 0.04%.
[ice-cold PBS/BSA 0.04%]
?. [Preparing cells for single cell analysis]
Centrifuge at 300 g for 5 min at 4 °C. Remove supernatant & re-suspend in 100 µL PBS/BSA 0.04%. Examine using hemocytometer with trypan blue.
[centrifuge at 300 g]
[re-suspend in ice-cold PBS/BSA 0.04%]
?. [Preparing cells for single cell analysis]
Adjust concentration to 1,000 cells/µL for Chromium or 100 cells/µL for DropSeq.
TITLE: Protein preparation for LC-MS/MS analysis
AUTHORS: Lihui Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is with regard to quantitative proteomics analysis to identify biomarkers of chronic myofascial pain and therapeutic targets of dry needling in a rat model of myofascial trigger points. For futher nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, the first step is to extract and prepare the protein of the sample.</div></div>

[STEPS]
?. The SDT lysis buffer was added to the sample and transferred to 2ml tubes with quartz sand consisting of 1/4-inch ceramic beads (MP 6540-424 for tissue samples).
?. The lysate was homogenized twice for 60 s using an MP homogenizer (24×2, 6.0M/S).
?. The homogenate was sonicated and then boiled for 15min.
?. After centrifuging at 14000 x g for 40min, the supernatant was filtered with 0.22µm filters. The filtrate was quantified with the BCA Protein Assay Kit (Bio-Rad Inc., Hercules, USA).
?. Protein samples of 20µg (each sample)were mixed with 5X loading buffer and the proteins were separated on 12.5% SDS-PAGE  (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) gel for 90min with a constant current of 14mA after boiling for 5min.
?. 200μg of proteins (each sample) were added to a sample of 30μl of SDT buffer (150mM Tris-HCl, 4% SDS, 100mM DTT, pH8.0) and then removed from the dithiothreitol (DTT), the detergent and other low-molecular-weight components, using uric acid buffer (150mM Tris-HCl, 8M urea, pH8.0) and 100μl iodoacetamide to block the reduced cysteine residues for Filter-aided sample preparation (FASP).
?. The samples were then incubated for 30min in the dark. Then, the filters were washed in triplicate with 100μl uric acid buffer and washed twice in100μl of 100mM tetraethyl-ammonium bromide (TEAB) buffer.
?. Finally, 4μg of trypsin in 40μl of TEAB buffer was used to digest the protein suspensions with overnight at 37°C.
?. Following sample digestion and immediately before use, the Tandem mass tag (TMT)  labeled reagents were equilibrated to room temperature, and 4-plex TMT reagent was applied to label 100μg of the peptide mixture of each sample, according to the instructions of the manufacturer (Thermo Fisher Scientific Inc., Waltham, USA).
?. The protein peptides were labeled with TMT-126, TMT-127, TMT-128, and TMT-129 for the CG, MG, DG and NDG groups for three biological replicates.
?. Incubation for 60min was undertaken to allow the reaction to occur, and 8μl of 5% hydroxylamine was added to the sample, which was incubated for a further 15 minutes.
?. TMT-labeled samples were fractionated into 10 fractions using a Pierce high pH reversed-phase fractionation kit by an increasing acetonitrile step-gradient elution.
?. Before LC-MS/MS analysis, the fractions were dissolved in 0.1% formic acid, according to the instructions of the manufacturer (Thermo Fisher Scientific Inc.).
TITLE: iBlot2--CHEM 584
AUTHORS: Ken Christensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Transferring your proteins from your SDS-PAGE gel to a nitrocellulose membrane for western blot is quick and straightforward.</div></div>

[STEPS]
?. [iBlot2 Transfer]
Watch the video about setting up and running your transfer to nitrocellulose at the following link: https://videos.thermofisher.com/detail/video/6059971760001/how-to-perform-a-western-blot-dry-transfer-using-the-invitrogen-iblot-2-dry-blotting-system
TITLE: DABCO Mounting Slide Protocol for Drosophila melanogaster embryos
AUTHORS: Ashley Albright

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for mounting Drosophila melanogaster embryos stored in PBS or PBT onto slides for conventional fluorescence and/or confocal microscopy. </div><div class = "text-block">Adapted from: 
</div><div class = "text-block">Kosman, D., Mizutani, C.M., Lemons, D., Cox, W.G., McGinnis, E., and Bier, E. (2004)</div><div class = "text-block"><a style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;:UNDERLINE;">https://science.sciencemag.org/content/305/5685/846.long</span></a></div></div>

[STEPS]
?. [Mount Slides]
Take off as much liquid as you can, resuspend embryos in an arbitrary amount of DABCO mountant (depends on how dilute you want the embryos to be, but generally a couple hundred ul). Allow the embryos to settle  to    at   or overnight at   before mounting. Embryos can be stored for months/years in microcentrifuge tubes in light-shielded boxes at .
?. [Mount Slides]
Transfer approximately  of embryos with using pipette tip cut with scissors onto a clean slide between two 22 x 22 mm coverslips approximately 0.5 cm apart in the middle of the slide. Cover with another 22 x 22 mm cover slip. Seal the edges with a quick-drying, transparent nail polish. Slides can be stored for a few weeks at   in the dark (or a few months at ) . Image source: https://www.uwo.ca/sci/research/biotron/pdf/microscopy/LSM_SampleGuide.pdf
35 µl
Keep in mind if you're DAPI staining, that tends to diffuse away. It is better to mount a fresh aliquot of embryos if samples are reanalyzed at a later date.
?. [Mount Slides]
Analyze embryos by conventional fluorescence or confocal microscopy.
?. [Make Mountant]
In light-shielded  conical (I cover mine in foil) add  of DABCO crystals, of 1X PBS, and  of glycerol.
50 ml
1 g
15 ml
35 ml
Read SDS for DABCO before starting.
?. [Make Mountant]
Mix on rocking platform until the solution is homogeneous. Store at .