eamag/protocols-with-steps huggingface dataset:
TITLE: Immunohistochemical labelling of spinal cord neurons involved in bladder activity
AUTHORS: Janet Keast, Peregrine Osborne, Nicole Wiedmann

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used for immunohistochemical visualisation of immediate early gene expression (c-Fos or Egr-1) in cryosections of rat lumbosacral spinal cord. Free-floating sections are processed in a double labelling protocol to distinguish immediate early gene expression in different neurochemical classes of spinal cord neurons:</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">ChAT [choline acetyltransferase]: preganglionic neurons</li><li style = "counter-reset:ol0;list-style-type:disc;">TH [tyrosine hydroxyls]: dopaminergic neurons</li><li style = "counter-reset:ol0;list-style-type:disc;">Pax2: inhibitory interneurons</li></ul></div></div>

[STEPS]
?. [Preparation of cryosections]
Cryoprotect fixed tissue (L5-S2 spinal cord) 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 (40 µm), collecting sections progressively across sets of 4 wells to collect 160 µm spaced series.
?. [Immunostaining]
Wash sections in PBS (3 x 10 min)
?. [Immunostaining]
Incubate sections in blocking solution at room temperature for 2 h
?. [Immunostaining]
Incubate sections in appropriate dilutions of primary antibodies (or combinations of primary antibodies) for 48-72h. Antibodies are diluted in PBS containing 0.1% sodium azide, 2% horse serum, and 0.5% triton-X.
?. [Immunostaining]
Wash sections in PBS (3 x 10 min)
?. [Immunostaining]
Incubate sections in appropriate dilutions of secondary antibodies (or combinations of secondary antibodies) 4 h in the dark. Antibodies are diluted in PBS containing 2% horse serum, and 0.5% triton-X.
A useful counterstain to visualise spinal regions can be included here, by adding NeuroTrace (fluorescent Nissl stain; 1:100) to the secondary antibody incubation.
?. [Immunostaining]
Wash sections in PBS (3 x 10 min)
?. [Immunostaining]
Mount sections onto glass slides and coverslip in preferred anti-fade mountant.
?. [Microscope]
Labeled neurons are counted and classified according to their immunoreactivity, including only nucleated neuronal profiles in the analysis.
For digital analysis, tile-scanning of complete spinal cord sections is recommended, ensuring that the order of sections (rostral to caudal) is noted.
TITLE: Optimum Max Keto Pills - Does It Have Side Effects?
AUTHORS: optimummaxpillseffects  

[DESCRIPTION]
Optimum Max Keto Pills - Does It Have Side Effects?


[STEPS]
1. Optimum Max Keto Pills - Does It Have Side Effects?
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Official Website:-https://marylandreporter.com/2022/04/25/optimal-max-keto-reviews-is-it-fraud-or-real/
TITLE: No Title
AUTHORS: 

[GUIDELINES]
<p><strong>STOCK SOLUTIONS:</strong><br /><br /> 1) 10.0 gm MgSO<sub>4</sub>·7H<sub>2</sub>O per liter d-H<sub>2</sub>O<br /><br /> 2) 1.0 gm KNO<sub>3</sub> per liter d-H<sub>2</sub>O<br /><br /> 3) 1.0 gm K<sub>2</sub>HPO<sub>4</sub> per liter d-H<sub>2</sub>O<br /><br /> 4) 50.0 gm disodium EDTA, 31.0 gm KOH per liter d-H<sub>2</sub>O<br /><br /> 5) 4.98 gm FeSO<sub>4</sub>·7H<sub>2</sub>O per liter acidified H<sub>2</sub>O (Acidified H<sub>2</sub>O is 999.0 mL d-H<sub>2</sub>O + 1.0 mL concentrated H<sub>2</sub>SO<sub>4</sub>)<br /><br /> 6) 11.42 gm H<sub>3</sub>BO<sub>3</sub> per liter d-H<sub>2</sub>O<br /><br /> 7) 8.82 gm ZnSO<sub>4</sub>·7H<sub>2</sub>O, 1.44 gm MnCl<sub>2</sub>·4H<sub>2</sub>O, 0.71 gm MoO<sub>3</sub>, 1.57 gm CuSO<sub>4</sub>·5H<sub>2</sub>O, and 0.49 gm CoNO<sub>3</sub>·6H<sub>2</sub>O per liter d-H<sub>2</sub>O<br /><br /><br /></p>

[STEPS]
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TITLE: Dissection and fixation of murine colonic tissue for myenteric plexus visualization
AUTHORS: Dante Heredia, Terence Smith

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Protocol for harvest of colonic intestinal tissue, with the intent of imaging the myenteric plexus.</div></div>

[STEPS]
?. A ventral midline incision is made and the whole colon is carefully excised into a Sylguard lined dissection dish.
?. Cut along the mesenteric border until the colonic tube is now a rectanular in shape.
?. Pin the colon at 110% of length and width mucosa side up. Gently remove the mucosal layer. Re-pin in a new Sylguard lined dish at 100% ensuring tissue is taught but not stretched.
?. Fix tissue using ice cold Zamboni fixative for 15 minutes.
?. Remove tissue from dish and wash 3 times in PBS for 15 minutes each wash.
?. Cut tissue into 3 sections; oral, middle and anal (in order to fit on slide) and mount on glass slide using Aquamount mounting media and a glass cover slip.
?. Image sample.
TITLE: Analysis of clinical features and early warning signs in patients with severe COVID-19: a retrospectivecohort study
AUTHORS: Xinpei Yue, Xinkui  Liu

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The Coronavirus disease 2019 (COVID-19) was first identified in Wuhan, China during December of 2019. Previous studies have described clinical aspectsof the COVID-19 illness;however, insufficient literature currently exists regarding detecting severe conditions in patients during the early diagnosisstage. Patients admitted to a designated hospital in the Henan Province of China with outcomes of either discharge or death before February 15, 2020 were enrolled retrospectively as a cohort study population. Patients who underwent at least one of the following treatments were considered a severe group: continuous renal replacement therapy (CRRT), high-flow oxygen absorption, noninvasive and invasive mechanical ventilation, or extracorporeal membrane oxygenation (ECMO); while,the remaining patients were considereda non-severe group. Data regarding basic information, initial symptoms, and first visit examination results were collected from electronic medical records (EMR) and compared between the severe and non-severe groups. Multivariate logistic regression analysis was performed to determine predictors. Areceiver operating characteristic (ROC) curve was applied to find threshold for each predictor. Altogether,104 patients were enrolled to our study with 30 and 74 patients in the severe and non-severe groups, respectively. A majority of older patients werein the severe group. In addition,patients in the severe group had higher direct bilirubin, lactate dehydrogenase, C-reactive protein (CRP), and procalcitonin along witha lower absolute lymphocyte value. Multivariate logistic analysis indicated that patients aged ≥63-years (odds ratio 41.0, 95% CI 2.8~592.4)，with an absolute lymphocyte value≤1.02×10^9/L (odds ratio 6.1, 95% CI 1.5~25.2), CRP≥65.08mg/L (odds ratio8.9, 95% CI 1.0~74.2) had more risk being diagnosed as severe condition.</div></div>

[STEPS]
?.
TITLE: Keto Advanced 1500 Review- Ingredients and Benefits, Price, Official Website -2022
AUTHORS: bawi.jaice  

[DESCRIPTION]
Different elements can cause weight gain, including an absence of satisfactory active work to consume calories. An individual ought to spend no less than 150 minutes out of every week on extraordinary activity and no less than two meetings devoted to expanding body strength. In any case, an enormous section of the populace has a stationary way of life and a great many people don't focus on actual work. Diet is a central point of contention, too. The majority of the populace's eating regimen comprises of a wealth of carbs and deficient solid fats and proteins. Accordingly, this diet makes fat stores around the body, making individuals become overweight.

[STEPS]
TITLE: Vandy - Mouse Myocardial Infarction
AUTHORS: Lin Zhong, Jeffrey Rottman, Chee Lim

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block">The most common cause of cardiovascular mortality in man is the outcome from myocardial infarction. Accordingly, it is necesarry to study the corresponding process of heart injury recovery in many mouse models relevant to human cardiovascular disease. This protocol describes the surgical induction of myocardial injury via 1) permanent ligation of a coronary artery in the heart (myocardial infarction), 2) cryoinjury, and 3) transient occlusion of a coronary artery followed by reperfusion (ischemia-reperfusion injury).</div></div>

[STEPS]
?. Mice are anesthetized with pentobarbital (50 mg.kg, IP).
?. The ventral neck and left parasternal region is shaved and disinfected with Betadine followed by 70% alcohol.
?. The mouse is positioned supineon a heating pad and a small incision is made through the skin underlying the trachea.
?. The trachea is exposed, a small puncture is made in the trachea, and endotracheal intubation is performed using a PE-50 tube.
?. The endotracheal tube is connected to a small rodent ventilator (Harvard Apparatus) for mechanical ventilation of the mouse.
?. ECG electrode leads are placed subcutaneously to monitor the ECG during myocardial infarction(MI).
?. With the use of a surgical microscope, a left thoracotomy is performed and the fourth intercostal space is entered using scissors and blunt dissection.
?. An 8-0 silk suture is placed through the myocardium into the anterolateral left ventricular wall around the left anterior descending (LAD) coronary artery.
?. MI: The suture is tied resulting in permanent ligation of the LAD and subsequent MI is confirmed via the use of the ECG monitor. Shams undergo the same procedure with the exception that the suture is not tied around the LAD, thus no ligation occurs.Ischemia-Reperfusion Injury: A sterile PE-50 tube is placed against the LAD and the suture is tied against the tube resulting in ligation of the LAD. During the ischemic period, the surgical area is covered with sterile gauze soaked in warm saline. Following the ischemic period, the tube is removed and the LAD ligature cut resulting in restoration of blood flow and reperfusion.Cryoinfarction: A small cryo-probe (3 mm diameter) is cooled in liquid nitrogen for 2 minutes and placed in a circumscribed region of the free LV wall (30 seconds) in order to achieve a reproducible transmural lesion.
?. The chest is closed in layers with 7-0 sutures.
?. The mouse is gradually weaned from the ventilator to resolve any possible pneumothorax.
?. Once spontaneous respiration resumes, the endothracheal tube is removed and the trachea incision is closed with 8-0 suture. The skin is then closed with 6-0 suture.
?. The mouseis maintained on the heating pad until fully recovered from anesthesia.
?. Buprenorphine is administered SC immediately following surgery and every 8-12 hr for 72 hr.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Zebrafish, and other small teleosts, are used as experimental models to evaluate human pathologies, including those linked to oxidative stress. The protocol presents an optimized technique to evaluate the activity of catalase, an important antioxidant enzyme, in zebrafish tissues, focusing on the brain and head kidney. The protocol is based on the classical Aebi (1984) method.</p>

[BEFORE_START]
<p>Every biochemical protocol needs to be validated in the laboratory when first introduced. The present protocol describes validation steps that were taken in LaNeC.</p>

[GUIDELINES]
<p>This protocol is intended to analyze catalase activity in zebrafish brain and head kidney tissue samples. It can be adapted for other fish species or other tissues without much hassle; however, tissue amounts need to be adjusted.</p>

[STEPS]
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TITLE: in vitro assembly and transformation
AUTHORS: Yuichiroh  Ikagawa

[DESCRIPTION]
For the in vitro assembly of the DNA fragments, we decided to use the NEBuilder®, an assembly kit based on the Gibson Assembly. In NEBuilder®, exonucleases break down the ends of the DNA fragments and hybridize the cohesive ends, followed by DNA polymerases that synthesize the broken strands. Finally, DNA ligase repairs the nick and completes the DNA assembly. it is expected that in vitro assembly will reliably transform the cyclized plasmid.

For the subsequent transformation, we used the NEB® 10-beta competent cell because NEB® 10-beta can be largely maintained the cloning efficiency even with high molecular weight plasmids, which we considered to be an advantage over other strains when transforming our team's plasmids.




[STEPS]
SECTION: in vitro assembly
3. DNA solutions and reagents were mixed according to the compositions in the table below.

 

SECTION: in vitro assembly
1. Thaw the DNA solution and NEBuilder® Assembly Master Mix on ice.
SECTION: in vitro assembly
2. Mix the DNA solution by vortexing, and centrifuge to collect the solution to the bottom of the tube.
SECTION: in vitro assembly
4. Incubate at 37℃ for 1 hour.
SECTION: transformation
5. Thaw Escherichia coli NEB® 10-beta competent cell on ice.
SECTION: transformation
6. Add 2 μl assembled sample into competent cell tube.
SECTION: transformation
7. Mix gently pipetting 4~5 times.
SECTION: transformation
8. Incubate on ice for 30 minutes.

SECTION: transformation
9. Heat shock at 42℃ for 30 seconds on heat block.

SECTION: transformation
10. Incubate on ice for 2 minutes.
SECTION: transformation
11. Add 950 μl of SOC medium and mix gently pipetting.
SECTION: transformation
12. Incubate the tube at 37℃ for 1 hour.
SECTION: transformation
13. Centrifuge at 5,000 g for 1 minute at room temperature
SECTION: transformation
14. Discard 900 μl of the supernatant.
SECTION: transformation
15. Resuspend cells with the remaining supernatant by Voltex
SECTION: transformation
16. Spread the whole culture on agar plates containing ampicillin.
SECTION: transformation
17. Incubate overnight at 37℃
TITLE: WAS IST Dieatovita und wie wirkt es?
AUTHORS: Eaharastcoumer  

[DESCRIPTION]
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[STEPS]
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TITLE: Genomic DNA prep with Quick Extract (QE) buffer 
AUTHORS: Hannah.Long  

[DESCRIPTION]
This protocol describes a high throughput extraction method to isolate genomic DNA for screening clones (e.g. from 24 well plate).

There are two options: i) you can split clones onto two plates and extract gDNA from one of those replica plates, or ii) during the process of splitting cells, you can keep some material for genotyping. This protocol describes the second option.

This protocol is for hESCs, but could work for other cell-types too.

[BEFORE_START]
Grow cells to around 50% confluency.

[STEPS]
1. Grow clones on a multi-well format, e.g. 24-well plate.
2. Prepare new matrigel coated plates for passaging clones and label PCR strip tubes corresponding to the position of the clones on the plates. E.g. 1-A1 would refer to plate 1, position A1 on the plate.
3. When clones are ready to be split, remove media and add ReLeSR for 30 s. Aspirate ReLeSR and leave in incubator for 3 min until colonies appear cracked.

5. Add appropriate amount of warmed mTeSR1 media for splitting, and tap plate gently. Using a P1000, gently scratch well to release remaining cells and put half media/cells in PCR tube and half in new plate.
4. In the meantime, prepare new plate by removing matrigel and adding some warmed mTeSR1  media.

6. Tap new plate to distribute cells and return to incubator. Monitor the following day and replace media (may see a lot of death).
7. Pulse spin strip tubes on bench-top mini-centrifuge. You may see pellet of cells. Remove media and replace with 50 µL  QuickExtract buffer. Tap to dislodge pellet. Could pipette up and down.

8. Place in thermocycler with the following settings:

65°C 8-10 min 
98°C 10 min
9. DNA samples are now ready to be used for PCR genotyping. For example, first try 1uL sample in a 10uL reaction (e.g. GXL PrimeStar - or Q5 polymerase). If PCR fails, try a dilution 1:5 of DNA, then 1:25 dilution etc.
TITLE: Open field test
AUTHORS: Chuyu Chen

[DESCRIPTION]
The open field test was developed to test locomotor activity.

[STEPS]
1. Mice were put in a 56 ×56 cm open-field arenas in noise-canceling boxes, illuminated by dim red lights
2. The 20-minute-long session started recoding when mice were placed in the center of the arena.
3. Locomotor activity was analyzed by the LimeLight 5 (Actimetrics) software and reported as distance traveled.
TITLE: Primer Design for Restriction Enzyme Cloning (E6901)
AUTHORS: New  England   Biolabs

[DESCRIPTION]
Guidelines for Primer Design for Restriction Enzyme Cloning (E6901).

[GUIDELINES]
Introduction

Appropriate restriction sites, absent in the target gene, are incorporated in the forward and reverse primers when a target gene is generated by PCR. The choice of the restriction site in the primers determines whether any, or which, extra amino acid residues will be attached to the terminus of the target protein after the cleavage of the intein tag.

Table 2 illustrates some examples of designing forward and reverse primers for pTXB1 and pTYB21. For cloning into pTXB1 one should clone a target gene between the NdeI (forward primer) and the SapI (reverse primer) sites in pTXB1. For the pTYB21 vector the SapI site can be used to clone the 5´ end of the target gene (PstI as the 3´ cloning site for pTYB21 is shown as an example for a reverse primer in the table). 
 

When constructing a N-terminal fusion (pTYB21) a stop codon should be encoded in the reverse primer. The reverse primer for the C-terminal fusion (pTXB1) should not include a stop codon. 

We recommend writing out your primers and cloning strategy in order to check SapI (or BspQI) digestion, the reading frames etc. For more information on cloning with SapI, please refer to: IMPACT FAQs

In general, more than 15 bp of target gene sequence is required for PCR (represented by 'NNNNNN...'). In Table 2 the restriction site is underlined (see in Construction of the Fusion Plasmid). The 'GGTGGT' sequence at the 5´ end of the primer is a random sequence of 6 bp to ensure efficient DNA cleavage by the restriction enzyme.

[STEPS]
TITLE: Immunoprecipitation protocol
AUTHORS: Chuyu Chen

[DESCRIPTION]
Immunoprecipitation procedure for brain tissue material

[STEPS]
1. Add 750ug tissue in 1ml IP buffer and antibody, incubate 4°C 2hrs on tube rotator
2. Prepare ProteinA/G beads 30ul/sample. Pool all beads in one tube to perform wash
3. Centrifuge 4°C 13000g 30sec, remove supernatant
4. Resuspend beads with 80ul PBS/sample. Centrifuge again at 13000g for 30sec.
5. Repeat wash 3 times. 
6. Remove supernatant with fine tip
7. Resuspend beads with 30ul PBS/sample
8. Add 30ul beads to protein sample
9. Incubate 4°C overnight on tube rotator
10. Centrifuge 4°C 4000rpm 4min, remove supernatant
11. Wash with IP buffer (+ protease inhibitor) 800ul/sample
12. Repeat 3 times, total 4 wash
13. Remove supernatant with fine tip
14. Add 1x loading buffer 25ul and 5ul reducing agent. 75°C 5min 
15. Centrifuge 4°C 4000rpm 4min, collect supernatant for western blotting
TITLE: Polymer-brushes-immersed-in-solvent-molecules
AUTHORS: Mike Edwards

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">By means of density functional theory (DFT), influence of solvent  molecules on polymer brushes is investigated. Osmotic pressure of  solvent molecules gives rise to a stronger stretching of the brush  chains in perpendicular direction. This suggests that the osmotic  pressure of solvent molecules is a driving force in increasing thickness  of brush layer.</div></div>

[STEPS]
TITLE: DNase I treatment in solution (after RNA extraction)
AUTHORS: Ben Kuipers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol used for the Dnase treatment after RNA isolation with the RNeasy mini kit. Protocol is based on the RNase-Free DNase Set and the RNeasy Mini Handbook 10/2013, Qiagen 'Appenix E: DNase Digestion of RNA before RNA Cleanup'.</div></div>

[STEPS]
?. DNase I Stock solution was made from the Promega Maxwell 16 LEV SimplyRNA kit DNase by adding 275 µL nuclease free water to the lyophilized DNase I.
?. Prepare a mastermix of 5 µL  Buffer RDD (RNase-Free DNase Set) + 1.25 µL DNase I stock (Promega Maxwell 16 LEV simplyRNA kit) + eluted sample. Make the volume up to 50 µL with RNase-free water (RNase-Free DNase Set).
?. Incubate the mixture on room temperature for 60 minutes.
?. Check the concentrations of DNA and RNA with Qubit.
?. Check the concentrations of DNA and RNA with Qubit again.