eamag/protocols-with-steps huggingface dataset:
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is the protocol for a large-scale Protein Expression Using BL21(DE3) Competent E. coli cells(C2527).

[BEFORE_START]
Determine the optimal time/temperature for the protein expression in a small scale trial.

[GUIDELINES]
<strong>BL21(DE3)&nbsp;Genotype:</strong><br />fhuA2 [lon] ompT gal (&lambda; DE3) [dcm] ∆hsdS<br />&lambda; DE3 = &lambda; sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7&nbsp;gene1) i21 ∆nin5<br /><br /><strong>NOTES:</strong><br />1. <strong>Caution:&nbsp;</strong>This product contains DMSO, a hazardous material. Review the MSDS before handling.<br /><br />2. <strong>Storage and Handling</strong>:&nbsp;Competent cells should be stored at -80&deg;C. Storage at -20&deg;C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80&deg;C, even if they do not thaw.

[STEPS]
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Paraffin sections of formalin-fixed human pancreas and isolated human islets were treated with 0.05% pronase for antigen retrieval, blocked with 2% bovine serum albumin (BSA; Sigma)/phosphate buffered saline (PBS), incubated overnight (4°C) with 10E4 (anti-HS) mAb (1/10; US Biological/Amsbio), washed and stained with AlexaFluor 488-goat anti-mouse IgM (Thermo Fisher). The same sections were washed, incubated with rabbit anti-human glucagon IgG (Abcam) or guinea-pig anti-pig insulin Ig (Dako), washed and stained with Alexafluor 568-donkey anti-rabbit IgG or AlexaFluor 568-goat anti-guinea-pig IgG (Thermo Fisher). The specificity of HS staining was checked on serial sections using IgMκ isotype control (BD Biosciences), instead of 10E4 mAb, together with anti-glucagon or anti-insulin antibody. Nuclei were stained with DAPI (0.2 μg/ml; Sigma). Sections were photographed using an automated Axio Observer inverted fluorescence microscope (Zeiss). Merged images were prepared using ZEN (version 2.3) software (Zeiss).</p>

[BEFORE_START]
<p>Materials:</p>
<p>       1.  Prepare graded alcohols and xylene for deparaffinizing tissue sections: 2 x xylene (250 ml/slide container), 2 x absolute ethanol (250 ml/slide container), 1 x 90% ethanol (250 ml), 1 x 70% ethanol (250 ml).</p>
<ol style="text-align: left;" start="2">
<li>Prepare 2% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). <br /><br /></li>
<li>Mabs and pAbs:<br />10E4 (anti-HS) mAb, Amsbio #370255-1<br />Goat anti mouse IgM AF488, Thermo Fisher #A21042<br />Polyclonal guinea pig anti-pig insulin, DAKO#A0564<br />Rabbit polyclonal anti-human glucagon, Abcam #ab133195<br />Goat anti-guinea pig IgG AF568, Thermo Fisher #A11075<br />Donkey anti-rabbit IgG AF568, Thermo Fisher #A10042<br />IgM<sub>κ</sub>, BD Biosciences #550340<br /><br /></li>
<li>Other reagents:<br />Pronase, Calbiochem #537088<br />Bovine serum albumin,3Sigma #A3294<br />DAPI, Sigma #D9524<br />ProLong Diamond Antifade Mountant, Thermo Fisher #P36961  <br /><br /></li>
</ol>

[GUIDELINES]
<p>10E4 anti-heparan sulfate (HS) mAb identifies highly sulfated HS localised in human beta cells but does not identify the less sulfated HS in alpha cells.</p>
<p> </p>
<p><u>Reference</u>:</p>
<p>Theodoraki A, Hu Y, Poopalasundaram S et al (2015) Mol Cell Endocrinol 399: 296-310.</p>

[STEPS]
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TITLE: RNAlater recipe
AUTHORS: Yin-Tse    Huang

[DESCRIPTION]
RNAlater recipe

[STEPS]
SECTION: Before you start
1. Submerge all your equipment and glassware in 1% bleach to get rid of nuclease
SECTION: Reagents
None. 0.5M Disodium dihydrate EDTA

 

SECTION: Reagents
None. 
SECTION: Before you start
None. Autoclave to deactivate DEPC
SECTION: Before you start
None. Keep the DEPC treated water at 4 °C 

SECTION: Reagents
2. DEPC treated water
1. Add 1 mL DEPC in 1 L ddH2O (0.1 % (v/v) ); MIX it well.
2. Let it sit for 1440 min at25 °C 
3. Autoclave it to deactivate DEPC 
4. Store the DEPC treated water at4 °C 

SECTION: Reagents
3. 0.5 M Disodium dihydrate EDTA:
1. Add 18.61 g disodium dihydrate EDTA in 100 mL ddH2O
2. Adjust pH to 8.0 with NaOH while stiring
SECTION: Reagents
4. 1 M tri-Sodium Citrate Dihydrate: 
Add 29.4 g tri-Sodium Citrate Dihydrate in 100 mL ddH2O, stir to dissolve
SECTION: Reagents
5. 1 M sulfuric acid: 
Add 5.33 mL sulfuric acid in100 mLddH2O, stir to dissolve
SECTION: Start to make RNAlater
6. Make a mixture of:
40 mL of 0.5 M EDTA + 25 mL 1 M tri-Sodium Citrate + 700 g Ammonium Sulfate +935 mL ddH2O; stir on a hot plate with low heat until ammonium sulfate dissolved

SECTION: Start to make RNAlater
7. Allow it to cool; adjust the pH of the solution to pH 5.2 using 1 M sulfuric acid

 

SECTION: Start to make RNAlater
8. The final concentration: 25 mM Sodium Citrate, 10 mM EDTA, 70 g ammonium sulfate in 100 ml ddH2O; pH 5.2.
SECTION: Start to make RNAlater
9. Transfer to screw top, nuclease-free bottles; label the date of make; store at 4 °C
TITLE: Bulk DNA Extraction (Ding Lab)
AUTHORS: Reyka Jayasinghe, Li Ding, Feng Chen, Satok

[DESCRIPTION]
Bulk DNA Extraction

[STEPS]
1. Cut tissue (~5 mg) and place in a 1.5 mL microcentrifuge tube. Add 180 µL Buffer ATL and 20 µL proteinase K, mix by vortexing and incubate at 56°C until completely lysed (1–3 h).
2. Add 2 µL RNase (100 mg/ml) and incubate for 30 min. at 37°C
3. Add 200 µL Buffer AL. Vortex for 15 sec.
4. Incubate at 70°C for 10 min. Briefly centrifuge the tube to remove drops from the lid.
5. Add 200 µL ethanol (96–100%). Vortex for 15 sec. Briefly centrifuge the tube to remove drops from the lid.
6. Pipet the mixture onto the QIAamp Mini spin column in a 2 mL collection tube. Centrifuge at 8,000 rpm for 1 min. at room temperature. Discard the flow-through and collection tube.
7. Place the QIAamp Mini spin column in a new 2 mL collection tube and add 500 µL Buffer AW1. Centrifuge at 8,000 rpm for 1 min. at room temperature. Discard the flow-through and collection tube.
8. Place the QIAamp Mini spin column in a new 2 mL collection tube and add 500 µL Buffer AW2. Centrifuge at 14,000 rpm for 3 min. at room temperature. Discard the flow-through and collection tube.
9. Place the QIAamp Mini spin column in a new 2 mL collection tube and centrifuge at full speed for 1 min. at room temperature.
10. Place the QIAamp Mini spin column in a new 1.5 mL microcentrifuge tube, add 50 µL DNase free water and incubate at room temperature for 1 min. Centrifuge 8,000 rpm for 1 min. at room temperature.
11. Repeat step 10 with a further 50 µL DNase free water.
14.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Intracavernous pressure (ICP) measurement is a well-established technique for assessing the erectile function, which was performed by cannulating either crus or shaft of the penis. But yet, as far as we know, no consensus has reached on the ES parameters of frequency and voltage response.</p>
<p>Moreover, the current technique for ICP measurement in the rat is difficult to perform, especially in the cannulation of the corpus cavernosum. It often leads to failure in the experiment and takes time to explore the optimum conditions for inexperienced investigators. In present study, we provided a detailed step-by-step procedure of ICP measurement.</p>

[STEPS]
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TITLE: Actinobacteria collection, enrichment and isolation
AUTHORS: Rene Flores Clavo, Cristian Daniel Asmat Ortega, Nataly Ruiz Quinones

[STEPS]
?. [Collection]
Collect samples aseptically in 600 ml screw-capped glass jar.
?. [Collection]
Pack the flasks in cool boxes with ice bags and transport them to the laboratory
?. [Collection]
Identify each sample by writing a code, sampling date, depth, sample color, pH, and sample location
?. [Enrichment and pre-treatment of samples]
Prepare 10 mL of Double Concentrated Sales Broth at pH 7,  in a beaker of 50 mL per sample.
?. [Enrichment and pre-treatment of samples]
Inoculate the broth with 10 mL of water and sediment from saline samples and take them to a water bath at 50 °C for 60 minutes to reduce bacterial contaminants load that is not of interest.
?. [Enrichment and pre-treatment of samples]
Incubate flasks at 28 °C for 7 days in aerobic conditions
?. [Enrichment and pre-treatment of samples]
Verify the growth of bacteria through microscopic observation.
?. [Enrichment and pre-treatment of samples]
Use plates with Modified Nutrient Agar at pH 7 for the isolation of actinomycetes
?. [Enrichment and pre-treatment of samples]
Culturing was carried out from the surface with apparent growth confirmed by microscopic observation
?. [Enrichment and pre-treatment of samples]
Incubate the plates at 28 °C for up to  30 days, evaluating the development of characteristic colonies and taking into account criteria such as macroscopic aspects of shape, size, elevation, consistency, and color of the colonies, also, microscopic features that characterize aerial filaments, substrate, as well as reproductive filaments and physicochemical characterization.
?. [Reactivation isolates]
Store the cryopreserved isolates in a refrigerated chamber at -20 °C
?. [Enrichment and pre-treatment of samples]
The isolates were preserved in 20% glycerol at -80 °C at the laboratory
?. [Reactivation isolates]
Culture the isolates in R2A broth supplemented with artificial seawater (ASW)  with 7% NaCl.
?. [Reactivation isolates]
Culture the isolates in Petri dishes containing Agar R2A with ASW supplemented with 7% NaCl and incubate the plates at 28 °C for 7 to 30 days.
?. [Reactivation isolates]
After Gram staining, observe under a microscope and stereoscope for macromorphological characterization.
?. [Reactivation isolates]
Preserve the isolates in 20% glycerol and keep them at -80 ° C for subsequent molecular identification studies.
?. [Enrichment and pre-treatment of samples]
Autoclave the broth in a glass flask, at 121°C and 15 psi of pressure for 20 min.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Quantitative measurements of both the copy number and spatial distribution of large fractions of the transcriptome in single-cells could revolutionize our understanding of a variety of cellular and tissue behaviors in both healthy and diseased states. Single-molecule Fluorescence In Situ Hybridization (smFISH)—an approach where individual RNAs are labeled with fluorescent probes and imaged in their native cellular and tissue context—provides both the copy number and spatial context of RNAs but has been limited in the number of RNA species that can be measured simultaneously. Here we describe Multiplexed Error Robust Fluorescence In Situ Hybridization (MERFISH), a massively parallelized form of smFISH that can image and identify hundreds to thousands of different RNA species simultaneously with high accuracy in individual cells in their native spatial context. We provide detailed protocols on all aspects of MERFISH, including probe design, data collection, and data analysis to allow interested laboratories to perform MERFISH measurements themselves.</p>
<p> </p>
<p>smFISH is a powerful technique because it allows the quantitative measurement of the exact copy number and spatial distribution of individual mRNAs within single cells in intact tissues. MERFISH extends these powerful abilities to the transcriptome scale by massively multiplexing smFISH measurements by encoding individual RNA species with error-robust barcodes and reading out the barcodes via a series of smFISH measurements. We anticipate this method to be relatively easily adopted by other laboratories and the techniques described in this collection should facilitate these efforts as will the supporting protocols and code that can be found at: </p>
<p><a href="http://zhuang.harvard.edu/merfish/ " target="_blank">http://zhuang.harvard.edu/merfish/</a>.</p>

[STEPS]
TITLE: immunofluorescent staining with anti-GFP and anti-CD63 antibodies
AUTHORS: rosanne.wouters, Peter Vangheluwe

[DESCRIPTION]
This protocol was used for immunofluorescent staining in fixed HeLa cells with anti-GFP and anti-CD63 antibodies, followed by confocal imaging.

[STEPS]
2. fix cells with 4% paraformaldehyde for 20 min at room temperature
3. permeabilize cells with 0.1% Triton X-100 in PBS for 5 min
4. block for 1 h with blocking buffer (PBS with 0.5% Tween20, 0.1% BSA, 0.2% FBS)
5. incubate coverslips with primary antibodies for 2 h at room temperature
(anti-CD63, exbio, 11-343-C100, mouse; anti-GFP, abcam, ab13970, chicken)
6. wash coverslips 3 times with PBS-T 
7. incubated coverslips 30 min with secondary antibodies 
(goat-anti-mouse-AlexaFluor647, goat-anti-chicken-AlexaFluor488)
8. wash coverslips 3 times with PBS-T 
9. incubate coverslips  with DAPI
10. wash coverslips 3 times with PBS-T 
11. mounte coverslips using  

12. images were acquired using an LSM780 con- focal microscope (Zeiss) with a 10x or 40× objective
13. colocalization analysis was performed with Fiji plugin Jacop
1. plate cells on glass coverslips and grown until they reach 60% conlfuency
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
The following protocol describes resuspension of lyophilized primers from (4) 96-Well plates shipped from Integrated DNA Technologies (IDT) at 3nmol per well using the epMotion 5075.

For information about ordering consult the Earth Micriobiome Projects website.

Barcoded primer constructs for 16S V4 region are also described in the Earth Microbiome Project website.

[BEFORE_START]
Please wear at least the minimum required personal protective equipment.
Ensure that all necessary kit components are available as well as user-supplied consumables.
Remove nuclease and nucleotide contamination from work surfaces and instruments prior to starting using an appropriate solution, such as RNase AWAY™ (Thermo Scientific™ catalogue: 700511), followed by wiping with 70% to 100% molecular biology grade ethanol to remove additional contaminants.

[STEPS]
SECTION: Prepare reagents
?. 
SECTION: Setup epMotion automation platform
?. 
SECTION: Execute automated protocol
?. 
SECTION: Prepare Plates
?. 
SECTION: Seal and store plates.
?.
TITLE: Lyophilized metal-antibody reconstitution
AUTHORS: Marc Bosse, Mike Angelo, Sean Bendall

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the procedure to prepare the reconstitution buffer and the resuspension of metal-labeled antibody.</div></div>

[STEPS]
?. [Stock solutions preparation]
Prepare Tris (10 mM), sodium azide (0.02%)
?. [Stock solutions preparation]
Add Tris 1M, pH 7.5 in  tube
500 µl
50 mL
?. [Stock solutions preparation]
Add  sodium azide ()
500 µl
?. [Stock solutions preparation]
Complete with 50 mL ultrapure water
?. [Stock solutions preparation]
Make aliquots of  and store a
10 mL
-20 °C
?. [Stock solutions preparation]
Prepare reconstitution buffer  ABCD1ReagentsFor 10 mLFor 20 mLFor 50 mL2Antibody stabilizer (Tris-based)3.757.518.753Tris (10 mM, pH 7.5), sodium azide (20mg/mL)6.2512.531.25
ABCD1ReagentsFor 10 mLFor 20 mLFor 50 mL2Antibody stabilizer (Tris-based)3.757.518.753Tris (10 mM, pH 7.5), sodium azide (20mg/mL)6.2512.531.25
?. [Stock solutions preparation]
For 10 mLAdd  of Antibody stabilizer (Tris-based)
3.75 mL
?. [Stock solutions preparation]
Add Tris (10 mM), sodium azide
6.25 mL
?. [Stock solutions preparation]
Filter using Milex-HV (0.45 µm) and syringe in new
?. [Stock solutions preparation]
Make aliquots of  and store at
1 mL
-20 °C
?. [Antibody suspension]
Take a aliquoted antibody tube and bring at
5 µg
0 Room temperature
?. [Antibody suspension]
Carefully open the screw cap tube
?. [Antibody suspension]
Add gradually 100 µL of reconstitution buffer over the lyophilized cake without touching it
?. [Antibody suspension]
Mix gently by flicking the bottom of the tube
?. [Antibody suspension]
Let it sit for at least at room temperature to allow rehydration
Highly recommended: store at    overnight to allow longer time for rehydration
?. [Antibody suspension]
Mix gently by flicking the bottom of the tube, while flicking inspect for translucid pellet and make sure that the lyophilized cake is totally suspended
?. [Antibody suspension]
Centrifuge: 10000 1324, 1 min
?. [Reconstitution label date]
Place the suspension dot label at bottom of the tube with the date recorded as MM/DD/YY; 50 µg/mL
?. [Reconstitution label date]
Store at 4˚C or use an aliquot immediately
TITLE: Yale - HDL Cholesterol
AUTHORS: John Stack, Gary Cline

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Procedure used to determine the concentration of HDL cholesterol in blood, serum, and plasma. HDL Cholesterol is determined in a two-step procedure. First chylomicrons, VLDL, and LDL are selectively reacted with cholesterol esterase and eliminated from the reaction. In the second step, the remaining HDL-cholesterol is assayed as described for total cholesterol.</div></div>

[STEPS]
?. Calibrate Cobas for HDL analysis by running a lipid calibrator, HDL Direct Reagent Reagent 1 and HDL Direct Reagent 2.
?. Sample handling as performed by the Cobas Mira Plus.  a) Pipette 3µL of sample into cuvette.  b) Add 180 µL of Direct Reagent 1.  c) Add 60 µL of Direct Reagent 2.  d) Mixture is incubated at 37ºC for 10 minutes.  e) Absorbance is measured at 600 nm.
TITLE: Pole Test
AUTHORS: Jhodi Webster

[DESCRIPTION]
This protocol describes the pole test, a simple and rapid behavioral assay used to evaluate motor coordination, balance, and bradykinesia (slowness of movement) in mice and rats. The test involves placing an animal head-up at the top of a vertical textured pole and measuring the time it takes to turn and descend to the base. It is highly sensitive to motor deficits caused by Parkinson's disease models, cerebellar lesions, or other neurological conditions, making it a valuable tool for phenotyping and drug screening.

[STEPS]
SECTION: Setup
1. Allow for one hour habituation period for mice in the testing room. 
SECTION: Setup
2. Place pole (approx 20.5in long, with rubber bands place at 1 inch intervals)
SECTION: Setup
3. Have an extra empty cage handy
SECTION: Training:
4. place mouse nose-down at the base of the pole and allow it to climb off onto the floor of the enclosure before being returned to home cage.  
SECTION: Training:
5. Do this twice for each mouse.  
SECTION: Probe Trials:
6. Place mouse nose-up at the top of the pole and allow for no more than 180 seconds to turn around and climb down to the bottom of the pole 
SECTION: Probe Trials:
7. Repeat this 5 times for each mouse. Allow for minimum of 30 seconds rest between each trial.  
SECTION: Measurements (in seconds):
8. Total descent time: from the time mouse was placed on the pole to the time taken for the mouse to reach to the bottom of pole.  
SECTION: Measurements (in seconds):
9. Turnaround time: time the mouse takes to turn around on the pole to face the bottom.
SECTION: Measurements (in seconds):
10. Time descending: total time to descend
SECTION: Considerations
11. If mouse pauses while turning around or descending, repeat trial.  
SECTION: Considerations
12. If mouse could not turn around, but instead descends with a lateral body position, then attribute total descent time to turnaround time.
SECTION: Considerations
13. If the animal immediately falls off the pole, assign the following max scores: total descent = 120secs, turnaround time = 120secs.
SECTION: Considerations
14. Do not allow mouse to climb over the top of the pole instead of making a turn. A small piece of cardboard can be placed at the top of the pole to prevent this.
TITLE: Cell counting
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Used for counting cells labeled with immunofluorescent markers in mouse brain sections. Sections should be stained, mounted, and imaged with high resolution (2048 x 2048 scanning).

[STEPS]
1. Using 2-3 sections to encompass the brain region of interest, acquire high resolution images (2048 x 2048) using tile scanning and z-stack acquisition.
2. Import images into ImageJ.
3. Apply maximum average projection and color adjust as needed, making sure to apply the same settings to all images.
4. For each image, use the multi-section tool in ImageJ to manually quantify the positively-labeled cells of interest. Multiple channels can be viewed to assess cells labeled with multiple markers.
5. Measure the region's area using the selection tool. Use this value to derive the relative density of cells.
TITLE: Descending Platform
AUTHORS: daniel.dautan daniel, Per Svenningsson

[DESCRIPTION]
Behavioral test to assess motor function.
The test is using a custom made 45-degree grid path with a width of 5cm and a length of 45cm. The 15cm one side is oriented with a 45-degree angle. The grid is formed by metal mesh with a 0.5cm space between.

[STEPS]
1. Place the custom-made, metal mesh grid to have an angle of 45 degrees with the floor.
4. At the end of the platform, place the animal cage to provide a safe target.
2. Place a camera above the grid to record the entire procedure. Here we use the Logitech C920 webcam and windows camera software.
 
11. Using the video, record the time for each mouse to go from:
1- hindlimb cross the starting spot line to 2- hindlimb touch the cage on the bottom.
12. Using 5 randomly selected frames of the period the mice descend the platform, measure the distance between the hindlimb of the mice, average the result to get usable values.
3. Orient the grid to have the starting spot horizontal.
5. Take the mouse out of its cage using a paper tube to avoid stress and gently place the mouse on the starting spot.
6. Allow the mouse to explore the starting spot for 2-3 minutes.
7. Gently push the mice towards the tilted part of the grid path.
8. As soon as the mouse's hindlimbs are crossing the separation between the starting spot and
the tilted spot, leave the mouse alone.
9. As soon as the mouse reaches the cage, stop the camera and place the cage back into the ventilated rack.
10.  Clean the platform with 50% ethanol solution to remove stress-related smells.
TITLE: Lysosomal activity DQ-Red BSA assay
AUTHORS: Razaul Karim

[DESCRIPTION]
This protocol details the Lysosomal activity DQ-Red BSA assay.

[STEPS]
SECTION: Cell Culture/Treatments:
1. 0.5x10^4/ml of cells plated on coverslips.
SECTION: Cell Culture/Treatments:
2. PFF-488 treatment.
SECTION: Cell Culture/Treatments:
3. PBS/trypsin (0.01%) wash.
SECTION: Cell Culture/Treatments:
4. DQ-BSA Red treatment: 

10 152790 min at 37 °C.
SECTION: Cell Culture/Treatments:
5. Fix cells using 4% PFA.
SECTION: Imaging:
6. Red fluorescence signal is detected only when fluorogenic substrate is hydrolyzed by lysosomal proteases.
SECTION: Imaging:
7. Leica Stellaries 8 confocal laser scanning microscope.
SECTION: Imaging:
8. Image analysis: 

Image J. quantification of the % area of the cell covered by DQ-Red signal divided by the number of cells (DAPI stained).
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is the protocol for the touchdown PCR assay</p>

[BEFORE_START]
<p>The assay has been optimized and validated for the Bio-rad C1000 Touch Thermocycler.</p>

[STEPS]
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