eamag/protocols-with-steps huggingface dataset:
TITLE: Oprah Winfrey Keto Gummies Reviews, Does It Real Or Fraud?
AUTHORS: keto-x-reviews  

[DESCRIPTION]
Oprah Winfrey Keto Gummies Reviews, Does It Real Or Fraud?

[STEPS]
SECTION: Oprah Winfrey Keto Gummies Reviews, Does It Real Or Fraud? Oprah Winfrey Keto – The Supplement for a Spare Shape and Slim Body!   There&#x27;s a superb product out then in the request that we all know by the name that goes as the Oprah Winfrey Keto. This blog is a complete in- depth analysis of this outstanding as well as vindicated weight loss product. Rotundity is the root issue of numerous health problems and will be removed completely from its core of it by the colorful rudiments.   In this trip, you shall lose down all your fat cells ever. Metabolism deteriorating sluggishly due to rotundity is common but not at all respectable. This brings in a cargo of problems related to your health for you which if not treated on time and at the foremost can frequently be fatal. The redundant weight now can be thrown down from the body.   Oprah Winfrey Keto has got a secret formula in it that effectively turns down rotundity and burns off each cell of your redundant quantum containing fats. These briskly consequences can get you that supremely stupendous fit body. As the name says this supplement is full evidence and complete in itself and you&#x27;re going to get a slim body presto when using this lozenge.  Oprah Winfrey Keto-what&#x27;s the each-new weight loss product?   Oprah Winfrey Ketosis now sitting at the top spot regarding a weight loss product and the stylish thing about it&#x27;s that this lozenge doesn&#x27;t let you lose your provocation by showing you small results each day so that you can get the feeling of negotiating your thing of weight loss sluggishly but surely on every day with this lozenge. This is the, sure enough, a way for your complete fat control.  This supplement known as the Oprah Winfrey Keto is been completely certified as well as laboratory vindicated as this product went through a lot ofcross-exams. Each set of deposited and redundant fats shall no longer be seen as those pounds will be naturally thrown off with its use. This is therefore the ultimate armament against the issue of rotundity. You&#x27;re now on the right path to attack stubborn fats.   How does the weight loss supplement work for the loss of weight?   The croakers call this product known as the Oprah Winfrey Keto as a universal bone because of the simple fact that it adapts and adjusts itself to different types and kinds of stoner’s bodies and in this way produces the quick ketosis and slimness results for you instantly that too without causing side goods. Then&#x27;s the detailed working way of the weight loss supplement.    Official Website:- https://urbanmatter.com/oprah-winfrey-keto-gummies-reviews-does-oprah-winfrey-acv-gummies-real/
1.
TITLE: Extraction Procedure
AUTHORS: Jing Xu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Dried leaves of </span><span style = "font-style:italic;">C. cyrtophyllum</span><span> (150 g) were weighted and sieved (20 mesh) in an herb disintegrator (118 Swing, Zhejiang, China); the powdered samples were extracted twice according to previous protocol [10].Solvent was removed from the combinedfiltrates and 61.44 g of ECE was obtainedand redissolved in distilled water (500 ml). The solution was partitioned with 3×250 ml petroleum ether(60–90 °C), 3×250 ml dichloromethane, 3×250 ml ethyl acetate and 3×250 ml </span><span style = "font-style:italic;">n</span><span>-butanol. The resulting extracts were concentrated to yield sub-fractions, PEF, DMF, EAF, BAF andRF, whichwere 065, 5.53, 4.13, 13.28 and 36.97 g, respectively. The samples were stored at 4 °C. </span></div></div>

[STEPS]
TITLE: Evaluation of seed morphology, seedling genetic variation, and components for seed storage of Agave landraces of commercial interest.
AUTHORS: Jesus A  Jimenez-Torres, Zurisadai Monroy-Gonzalez, Juana Juarez-Muñoz

[DESCRIPTION]
Sexual propagation of Agave plants is an incipient cultivation method, these plants withstand drought and adverse growing conditions; therefore, research on Agave’s diversity, seed processing and storage, could support its cultivation on marginal lands. The aim of this work was to evaluate seed morphology, germination, and seedling genetic diversity of six seed origins (species*provenances) of Agaveplantscollected in five provenances from Mexico. Seed longevity was evaluated in two seed origins after a ten-year storage period. Seed morphology and seedling genetic variation results demonstrated intra- and interspecific variation within Agave salmiana and the other seed origins, respectively. After a ten-year storage period seed germination of two A. salmiana seed origins remained relatively stable, storage conditions and seed variables of this work can serve as reference parameters for future analyses. To the best authors’ knowledge this is the first report of Agave’s seed longevity evaluation after a ten-year storage period.

[STEPS]
TITLE: Standard PCR protocol
AUTHORS: Victoria Jackson

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Generic PCR protocol</div></div>

[STEPS]
?. [Master mix]
For a  reaction volume use the following recipe: dH2O buffer MgCl2 FWD primer (from a   working solution) REV primer (from a   working solution) dNTPs (from a   mix) Taq polymerase
25 µl
14.9 µl
5 µl
1.5 µl
1 µl
1 µl
0.5 µl
0.1 µl
?. [Master mix]
Prepare the master mix   allowing for 10% pipetting error (eg. if carrying out 10 reactions, calculate the master mix quantities needed for 11 reactions).
on ice
Remember to include a negative control with no DNA template and, if possible, a positive control using a known DNA template.
?. [Preparing PCR reaction tubes]
Aliquot  of master mix into each 0.2 mL PCR tube.
24 µl
?. [Preparing PCR reaction tubes]
Add  of either DNA template, dH2O (negative control) or known DNA (positive control) to the PCR tubes and place in a thermocycler.
1 µl
?. [Thermocycling program]
Standard PCR thermocycler program (30 cycles):
?. [Thermocycling program]
Initial denaturation at  for
94 °C
?. [Thermocycling program]
Denature at  for
94 °C
?. [Thermocycling program]
Annealing at ~ (depending on primers) for
55 °C
?. [Thermocycling program]
Extension at  for  x 29
72 °C
?. [Thermocycling program]
Final extension at  for
72 °C
?. [Storage]
Store PCR products at , or at  for long term storage.
4 °C
-20 °C
?. [Analysis]
Check if the PCR was successful by gel electrophoresis.
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TITLE: ProExM
AUTHORS: monica.fernandez-monreal

[DESCRIPTION]
Classical protocol of protein pre-labeling Expansion- ProExM
(c) Ed Boyden

[STEPS]
SECTION: Solutions
1. Gel and disgestion
SECTION: Solutions
2. Fixation
SECTION: Fluorescence Labeling
3. Fixation: Use fresh 4% PFA solution in PBS (w/ or wo/ 4% sucrose) for 15-20 min. Wash 3 times in PBS and quench aldehydes with glycine or sodium borate.
SECTION: Fluorescence Labeling
4. Permeabilize in blocking buffer (0.2% fish gelatin, 0.1% or 0.3% triton in PBS), 1 hr at RT.
SECTION: Fluorescence Labeling
5. Incubate with primary antibodies in blocking buffer 1h at RT, or overnight at 4ºC.
SECTION: Fluorescence Labeling
6. Wash slices with blocking buffer, 4 times, ~10 min each.
SECTION: Fluorescence Labeling
7. Incubate slice with secondary antibodies in blocking buffer 1 h at RT on a shaker.
SECTION: Fluorescence Labeling
8. Wash with PBS, 3 times, ~10 min each.
SECTION: Anchoring
9. Dilute AcX 1:100 (0.1 mg/mL) in 1x PBS (prepare 500 uL/well for 12-well plates).
11. Treat stained expressing genetically encoded FPs slices or coverslips for > 6 hours at RT (this reaction can be left overnight).
10. Wash 2x 15 minutes with PBS before proceeding to gelation. Samples can be stored at 4º C.
SECTION: Gelling
12. Make sure to remove excess PBS from brain slices before incubation with gelling solution. Incubate slices in gelling solution in a 24-well plate for 25 min at 4C. Coverslips with cells can be mounted directly.
SECTION: Gelling
13. Create a humid gel chamber. For 50 um slices, 2 coverglass were used to create a 170 um-depth sandwich chamber. Cell cultures were mounted cells side-down over a drop of gel (35 um for a coverslip of 12 mm). Make sure the slices are flat, and avoid air bubbles trapped inside the chamber.
SECTION: Gelling
14. Incubate 1h or 2h at 37ºC.
SECTION: Digestion and expansion
15. Cover the gel in digesting buffer for 15 min and add proteinase K for and overnight incubation @ room temperature (make sure at least 10-fold excess volume of digestion buffer is used, and make sure make it does not dry out).
SECTION: Digestion and expansion
16. Wash slices with excess volume of ddH2O (we usually use at least 10x the final gel volume), 3-5 times, for 15mins each time. Slice expansion reaches plateau after about the 3rd or 4th wash. The expansion chamber needs to be of adequate size for the sample: hemislices of mouse brains fit nicely in a 6-well plate when the excess gel around the brain is trimmed away (a razor blade works well for this). 18 mm coverslips can be punched in 4 mm medals before expansion to get gels of around 16 mm.
SECTION: Mounting the gel
17. Use a cleaned coverslip and add poly-L-lysine 0.1% solution to the top surface for 20-30 min. Rinse with ddH2O and air dry. 
SECTION: Mounting the gel
18. Remove excess of liquid around the gel and place the expanded sample on the coverslip. After 20-30 sec, add ddH2O to keep the sample hydrated.
TITLE: Lysosome proteolysis analysis with DQ-BSA
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Protocol to analyze lysosomal proteolysis in astrocytes generated from iPSCs.

[STEPS]
1. Coat a 96 well plate Greiner 96 well Microplates Black with gelatin 0.1% or Geltrex LDEV Free hESC Quality 5 ml Thermo Fisher Scientific Catalog #A1413302 1:100


2. DIssociate  extracted astrocytes with   for 5 min  at 25 °C 

3. 300 x g, 3 min, 25 °C 

4. Resuspend the pellet in 1 mL of astrocyte media (https://www.sciencellonline.com/astrocyte-medium.html).

5. Count cells and plate 10k cell per well in 100 µL  of astrocyte media.

6. Change the media to mature astrocyte media media in the next day.
7. On the 3rd day, remove the media incubate with  diluted  in mature astrocyte media for  45 min 

8. Remove the media
9. Wash with PBS 3 times.
10. Add 100 µL  of mature astrocyte media with vehicle or with  Bafilomycin A1 100 nanomolar (nM) 

11. Proceed to image in the Incucyte® SX5 Live-Cell Analysis System for 1260 min  using the red channel
TITLE: Immunohistochemistry (neural organoids)
AUTHORS: anita.adami

[DESCRIPTION]
This protocols describes how to perform immunohistochemistry on neural organoids

[STEPS]
SECTION: Fixing and mounting
1. The organoids were fixed in 4% paraformaldehyde for 120 min at Room temperature .  
SECTION: Fixing and mounting
2. They were subsequently washed three times with KPBS and left in a 1:1 OCT:30% sucrose solution and OCT (HistoLab) mixture 120 min.
SECTION: Fixing and mounting
3. The organoids were then transferred to a cryomold containing OCT and frozen on dry ice and stored at -80°C. 
SECTION: Sectioning
4. Prior to the staining protocol, the organoids were cryosectioned at a thickness of 20 μM. 
SECTION: Staining
5. For immunohistochemistry, sections were washed in PBS1X for 10 min and then blocked and permeabilized in Blocking solution (0.3% Triton X-100 and 4% normal donkey serum in PBS1X) for at least 60 min. 
SECTION: Staining
6. The sections were then incubated with primary antibodies in blocking solution at 4 °C 60 min.

The primary antibodies used for neural organoids characterisation were rabbit anti-PAX6 (1:300; BioLegend, Cat# 3700, RRID:AB_2242334)) and rat anti-ZO1 NB110-68140, RRID:AB_1111431(1:300; Novus)).
SECTION: Staining
7. After incubation with the primary antibodies, the sections were incubated for 1 h with the appropriate secondary antibodies (Alexa Fluor 488, 594, 647 used at 1:400; Molecular Probes).
SECTION: Mounting
8. Finally, the sections were mounted on gelatin-coated slides and coverslipped with PVA-DABCO containing DAPI (1:1000).
TITLE: Indirect ELISA for detection of anti-HIV antibodies in cats immunized with a HIV gp120 peptide.
AUTHORS: Angel  Justiz-Vaillant

[STEPS]
?. The ELISA describes detection of antibodies against HIV proteins. It is an ELISA with a modification. The plate is coated with HIV peptides, fragment 254-274 of gp120.
?. The modification is that triplicates of 1/16 dilutions of cat sera in PBS non-fat milk are added to the micro-wells.
?. After incubation for 90 min at RT the microplate is washed 4X using PBS-Tween 20.
?. Then,  25 µl of recombinant protein LA-HRP diluted 1:1000 (Sigma-Aldrich) is added to the assay.
?. The microplate is incubated for 1h at RT and then, it is washed four times.
?. Tetramethylbenzidine (TMB) solution (50 μl) is added to each well. .
?. After a further incubation of 15 min in the dark, the reaction is stopped.
?. The microplate is read at 450 nm in a microplate reader at 450 nm. Geometric mean antibody titer is calculated using Perkins method.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This protocol is for purification of Cas9::NLS<sub>SV40</sub>::His<sub>6 </sub>from:<br /><span class="cit-title"><span class="cit-auth cit-auth-type-author">Paix A</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-auth cit-auth-type-author"> Folkmann A</span><span class="cit-sep cit-sep-separator">,</span><span class="cit-auth cit-auth-type-author"> Rasoloson D</span><span class="cit-sep cit-sep-separator">, </span> <span class="cit-sep cit-sep-last-separator">and</span><span class="cit-auth cit-auth-type-author"> Seydoux G (2015) <a href="http://www.genetics.org/content/early/2015/07/17/genetics.115.179382.abstract" target="_blank">High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR/Cas9 Ribonucleoprotein Complexes</a></span>.</span>  Genetics <span class="cit-elocation">genetics.115.179382</span><span class="cit-sep cit-sep-after-article-pages">; </span><span class="cit-ahead-of-print-date"><span class="cit-sep cit-sep-before-article-ahead-of-print-date">Early online </span>July 17, 2015. <span class="cit-doi"><span class="cit-sep cit-sep-before-article-doi">doi:</span>10.1534/genetics.115.179382<br /><br /></span></span>Please see the <a href="http://www.genetics.org/content/early/2015/07/17/genetics.115.179382.abstract" target="_blank">full manuscript</a> for detail.

[GUIDELINES]
Protocol updates will be posted on the <a href="http://www.bs.jhmi.edu/MBG/SeydouxLab/" target="_blank">Seydoux lab website</a>.<br />Main protocol: <a href="https://www.protocols.io/view/Direct-delivery-CRISPR-HDR-editing-protocol-for-C-dri54d" target="_blank">Direct delivery CRISPR-HDR editing protocol for <em>C. elegans</em></a> <br /><br /><strong>Buffers</strong>: <br />Buffer A: 20mM Tris ph 8.0, 250 mM KCl, 20 mM imidazole, 10% glycerol, 1 mM TCEP <br />Buffer B: 20mM Tris ph 8.0, 800 mM KCl, 20 mM imidazole, 10% glycerol, 1mM TCEP <br />Buffer C: 20mM Hepes ph 8.0, 500 mM KCl, 250 mM imidazole, 10% glycerol <br />Buffer D: 20mM Hepes ph 8.0, 500 mM KCl, 20% glycerol<br /><br /><strong>Purified Cas9::NLS<sub>SV40</sub>::His<sub>6</sub> resolved by SDS-PAGE:<br /><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/4bcz36.png" alt="" data-src="https://s3.amazonaws.com/pr-journal/4bcz36.png" data-ofn="sdspage.png" /><br /></strong>Recombinant Cas9::NLS<sub>SV40</sub>::His<sub>6</sub> was affinity purified using Niâagarose (lane 1). Pooled eluent was flowed over Q sepharose to remove contaminating DNA bound to Cas9 (lane 2). Samples were resolved by SDSâPAGE and visualized by coomassie staining.Â Â <br /><br /><strong>Cas9 activity assay</strong>: We recommend testing your Cas9 preparation using <a href="https://www.protocols.io/view/Seydoux-lab-Cas9-preparation-test-Tag-gtbp-1-with-duj6um" target="_blank">this method</a>.

[STEPS]
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?.
TITLE: Cryosectioning mouse brain
AUTHORS: Pranay  Srivastava, Waijiao Cai, Xiqun Chen

[DESCRIPTION]
This protocol details the cryosectioning of mouse brain.

[STEPS]
SECTION: Cryosectioning mouse brain
1. Place the mouse brain in the mouse brain slicer (on ice). Cut the hinder of the brain and put it in 4% PFA   at 4 °C.

SECTION: Cryosectioning mouse brain
2. Change the solution to 30% sucrose/PBS at 4 °C until the brain sinks in the bottom ( ).

SECTION: Cryosectioning mouse brain
3. Change the solution to 60% sucrose/PBS at 4 °C for 2880 min-7200 min (optional).

SECTION: Cryosectioning mouse brain
4. Cut 1/2 to 2/3 of the cerebellum to make a flat seat.
SECTION: Cryosectioning mouse brain
5. Cut the pieces of foil for the tissue wrapping and label the pieces with chemical resistant marker.
SECTION: Cryosectioning mouse brain
6. Place a small beaker containing 2/3 2-methylbutane, at least half buried in dry ice. Allow it to cool enough (when you see it turn white and thick at the bottom of the container).
SECTION: Cryosectioning mouse brain
7. Gently place the mouse brain into 2-methylbutane and allow it to sit for a while until it no longer “smoking” (~20 s).

SECTION: Cryosectioning mouse brain
8. Place a layer of OCT on the foil on dry ice. Wait a few seconds until OCT turns to half opaque.
SECTION: Cryosectioning mouse brain
9. Place the brain on the layer of OCT and place another layer of OCT to cover the brain.
SECTION: Cryosectioning mouse brain
10. Wrap the brain with the labeled foil and store the brain at -80 °C until sectioning.

SECTION: Cryosectioning mouse brain
11. Adjust the cryostat to: chamber -22 °C, specimen-23 °C, 30 μm.

SECTION: Cryosectioning mouse brain
12. Place the brains in cryostat when it reaches -22 °C for half hour to allow the temperature balanced.

SECTION: Cryosectioning mouse brain
13. In 12-wells plate, add 1.5 mL cryo-protective solution (30% Glycerol, 30% Ethylene Glycol in PBS) per well.

SECTION: Cryosectioning mouse brain
14. For stereological dopaminergic neurons counting, start to collect the sections when it close to substantial nigral (midbrain is apple-ish like shape. Hippocampus spread to 2/3 of the cortex). Collect ~10-12 sections per well. Store plates at -20 °C until staining.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This is a collection of G-Biosciences MegaLong™ protocols for the isolation of &gt;100kb Genomic DNA .</p>
<p> </p>
<p>MegaLong™ isolates high molecular weight (&gt;100kb) genomic DNA from a variety of samples, including animal tissues, cultured cells, whole blood, bacterial and yeast. Please refer to the appropriate protocol below, depending on your application.</p>

[STEPS]
?.
TITLE: Making low peptone NGM for imaging plates
AUTHORS: Ida Barlow, Priota Islam

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">C. elegans is maintained in the laboratory on Nematode Growth Medium (NGM) agar which has been aseptically poured into petri plates. The NGM agar medium can be poured into petri plates easily and aseptically using a peristaltic pump. This pump can be adjusted so that a constant amount of NGM agar is dispensed into each petri plate. A constant amount of agar in the plates reduces the need for refocusing the microscope when you switch from one plate to another.</div><div class = "text-block">For imaging purposes, low peptone NGM is prepared which contains less concentration of bactopeptone as compared to the normal NGM. This is because we don't want the seeded bacteria to grow too much and obscure the images, rather just sufficient enough to keep the worms from crawling out of the plate due to lack of food. The imaging plates can be 35mm, 60mm or 90mm in diameter depending on the assay design.</div></div>

[STEPS]
?. [Pre-Autoclave:]
Book the autoclave (notebook on top of the machine). Take clean flasks from the glass kitchen. Measure all the pre-autoclave reagents and add to the flask (Use a new weighing boat and spatula for each reagent. Also, the cholesterol is kept in the fridge.) Once water is added mix thoroughly and label with autoclave tape (‘Low peptone NGM Rm 5020’). Makesure the bottle is not screwed completely when placing it inside the autoclave machine.
?. [Using the autoclave:]
- Turn ON the autoclave     - Make sure that the autoclave’s probe bottle is the same size as the largest bottle you use and fill it with water.      - Place the temperature probe in it.     - Fill up the autoclave with water until it reaches the grill.     - Place the bottles in the autoclave and make sure that the cap is not screwed completely.     - Check the waste flask is not too full     - Use ‘media’ program.      - Press START.     - It will take about 2 hours for a 500ml bottle to autoclave and about 2.5 hours for 1L or larger bottles.
?. [Post autoclave:]
When autoclave is complete, remove the probe flask  Make sure to wear gloves as the flask will be hot Let the agar to cool to around 55°C, ie the bottle is cool enough to hold for a second with a gloved hand. Add the post autoclave reagents. Mix it well and start pouring onto imaging plates   (See Protocol for plate pouring)Try not to shake the bottle too much while mixing to avoid air bubbles.The agar needs to be warm to be poured without blocking the tubings, so try to pour as quickly as possible and if not poured immediately put the bottle on a waterbath set to 60C until being used.
TITLE: SEGA protocol collection
AUTHORS:  Carolyn Bayer, Maja Rennig, Anja  Ehrmann, Morten Norholm

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">SEGA, the Standardized Genome Engineering Architecture, is a comprehensive strain collection that enables genome engineering by combining only two reagents: a DNA fragment that can be ordered from a commercial vendor and a stock solution of bacterial cells followed by incubation on agar plates. Recombinant genomes are identified by visual inspection using green-white colony screening akin to classical blue-white screening for recombinant plasmids. The modular nature of SEGA allows precise multi-level control of transcriptional, translational, and post-translational regulation. The SEGA architecture simultaneously supports increased standardization of genetic designs and a broad application range by utilizing well-characterized parts optimized for robust performance in the context of the bacterial genome.</div><br/><div class = "text-block">This protocol is a collection of all important SEGA protocols.</div></div>

[STEPS]
TITLE: FFPE Tissue Sectioning for Staining
AUTHORS: Marda Jorgensen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Method for sectioning paraffin blocks for staining </div></div>

[STEPS]
?. Prepare a water bath at 42°C and place it next to the Microtome.
?. Insert a new blade for sectioning each block
?. Section the tissue between 4-5 µm thick.
?. Place the sectioned tissue in the water bath for a few seconds and observe it expanding.
?. When the observed expansion is enough to remove folds and wrinkles from the tissue,  quickly place positive charged microscope slide in the water bath and gently move it upward towards the tissue.
?. Doing so, the tissue will lay on the slide as this is moved out from the water bath.
?. Put the slides upright in a rack to let air dry overnight or dry in 50°C oven for 1 hour.
?. Label positive charged microscopes slides.