eamag/protocols-with-steps huggingface dataset:
TITLE: Astrocyte extraction from brain organoids
AUTHORS: gustavo.parfitt

[DESCRIPTION]
Protocol for astrocyte extraction from brain organoids.  

[STEPS]
2. Collect 10-20 spheres and place in a 6 well plate  (day 40+ spheres)

1. Coat a 6 well plate coat with gelatin 0.1% or   1:100

3. Wash in PBS twice 

4. Aspirate the  PBS
5. Add 1 mL  of  for  5 min 

6. Triturate using glass a pipette (2-3 up and down)

7. Transfer the cells and tissue (even the chunks, the trituration is more effective if you have chunks of tissue) to the coated 6 well plate 
8. Aspirate the extra TrypLe
9. Add 3 mL of astrocyte media (https://www.sciencellonline.com/astrocyte-medium.html)

10. Half media change media every 3 days or until it turns orange.
11. Keep changing media every 3d until you observe good amount of cell attached to the plate (may vary according to the patterning)
12. Passage the astrocytes to a 15 cm plate in Astrocyte media once they reach 80% confluency
13. For the experimental protocols after passage cell will be incubated in maturation media (TBD)
TITLE: Chairside vs Labside All-Ceramic FDPs - A Systematic Review 
AUTHORS: tim.joda

[DESCRIPTION]
Nowadays, the term digital is ubiquitous and everything has to be digitally branded to be up to date. Digitalization is not a trend, it is reality – and dental medicine is no exception. Dentistry is digital, the attribute phrasedigital dentistryis no longer necessary.
This continuous IT process has opened up new possibilities in reconstructive dentistry. On the one hand, digital 3D diagnostics has rapidly developed to the point of treatment simulation using virtual dental patients, and on the other hand, new production processes using computer-aided design / computer-aided manufacturing [CAD/CAM] for therapy with monolithic restorations are now feasible.
Chairside dental protocols allow in-office treatment of prosthetic restorations during a single appointment: (1) starting with clinical preparation of the restoration site, either tooth-borne or implant-retained; (2) followed by intraoral optical scanning [IOS]; (3) further CAD/CAM processing of the dental restoration; and finally (4) clinical insertion.
However, chairside does not necessarily mean a ‘one-man-show’ by the clinician, but rather a treatment option for immediate rehabilitation on a single-visit basis. Patients expect high esthetics, prosthetic invisibility, and incorporated function in harmony. They are also looking for a time-saving solution and convenient treatment experience, but they do not care who performs and finalizes the technical fabrication of the restoration. In the end, the prosthetic reconstruction must meet all expectations for both the patient and the dentist.

[STEPS]
1. SYSTEMATIC REVIEW
Chairside versus labside CAD/CAM monolithic all-ceramic fixed dental prostheses (FDPs): A systematic review
2.  
Focused question (PICO)Do chairside versus labside CAD/CAM monolithic all-ceramic fixed dental prostheses demonstrate comparable outcomes in terms of cost-effectiveness, patient-reported outcome measures, esthetics, and survival and success after 1 year in function?Search StrategyPopulationMonolithic all-ceramic fixed dental prostheses (FDPs)[single crowns and multi-unit restorations] InterventionChairside CAD/CAM workflows ComparisonLabside CAD/CAM workflows Outcome(s)Cost-effectiveness (economics as time-efficiency and costs)*Patient-reported outcome measures (PROMs) [2nd]Esthetics [2nd]Precision and accuracy [2nd]Technical and biological complications [2nd]Survival and success (follow-up > 1 year) [2nd]
 
3.
TITLE: Protocols for "Chromosome-level genome assembly of the humpback puffer, Tetraodon palembangensis"
AUTHORS: Rui Zhang, Chang Li, Mengjun Yu, Xiaoyun Huang, 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>In this study we assembled the first chromosome-level genome assembly of the humpback puffer (</span><span style = "font-style:italic;">Tetraodon palembangensis</span><span>), a species of poisonous freshwater pufferfish mainly distributed in Southeast Asia. This utilized Oxford Nanopore and BGISEQ-500 sequencing, stLFR libraries and Hi-C assembly, and the protocols for nucleic acid extraction, library construction and sequencing are all made available here. The resulting genome size was 362 Mb with a ~1.78 Mb contig N50 and ~15.8 Mb scaffold N50. Based on the assembled genome, ~61.5Mb (18.11%) repeat sequences were identified, 19,925 genes were annotated, and 90.01% of these genes could be predicted with function. Finally, a phylogenetic tree of ten teleost fish species was successfully constructed using this and public data. These protocols are made available for others carrying vertebrate genome assembly, and the open data and published study for comparative genome studies.</span></div></div>

[STEPS]
TITLE: Cryoprotection of Mouse Brain
AUTHORS: Asta Zane, Nicole J Corbin-Stein, Gabrielle Childers, Jhodi Webster, Vickie Yang, Woong-Jai Won, Rajesh Gupta, Ashley Harms

[DESCRIPTION]
This protocol allows for accurate cryoprotection of mouse brain, post-perfusions, to be used for histology. The methods utilize another PFA fixation step followed by sucrose incubation. 

[STEPS]
SECTION: PROCEDURE
1. Transfer the brain into 5-10ml of 4% PFA solution in PBS for 2 hours at room temperature in a 15 ml conical tube.
SECTION: PROCEDURE
2. Transfer the brain into 30% sucrose solution in PBS, wait until it sinks to the bottom for 48-72 hours at 4°C. 
SECTION: PROCEDURE
3. Freeze brain on dry ice and store at minus -80 °C till sectioning.   
SECTION: PROCEDURE
4. To freeze the brain cut foil into pieces that will be labeled accordingly. Lay the foil on the top of the dry ice. Put the brain on the foil. Let the brain to freeze. It should change its color to white, and become hard. Wrap the foils edges. Put samples in the box, label the box with your project, and put it to minus -80 °C.
TITLE: No Title
AUTHORS: 

[GUIDELINES]
To suppress both positive and negative phototactic behavior, we use an aversive stimulus. We place a filter paper wetted with either a 0.15M quinine/water solution (gustatory stimulus) or an 0.1 mixture (v/v) of benzaldehyde/paraffin oil (olfactory stimulus) in the preferred tube, i.e., the transparent or the opaque tube, depending on the flight status of the flies and hence their phototaxis (positive or negative, respectively).

[STEPS]
?. 
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TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Deep learning is an emerging field that promises unparalleled results on many data analysis problems. We show the success offered by such techniques when applied to the challenging problem of image-based plant phenotyping, and demonstrate state-of-the-art results for root and shoot feature identification and localisation. We use fully automated trait identification using deep learning to identify quantitative trait loci in root architecture datasets. The majority of (12 out of 14) manuallyidentified QTL were also discovered using our automated approach based on the deep learning detection to locate plant features. We predict a paradigm shift in image-based phenotyping bought<br />about by deep learning approaches.</p>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Whole Mouse Brain Delipidation - Dichloromethane
AUTHORS: Holly Myers, daphne.toglia

[DESCRIPTION]
This protocol describes the delipidation of a mouse brain specimen using a modified iDISCO protocol. The brain is dehydrated with methanol and then cleared of lipids with dichloromethane to prepare for refractive index matching (see protocol Refractive Index Matching - Ethyl Cinnamate). This will allow for viewing and imaging of internal brain structures with a light sheet microscope. This technique does not preserve endogenous fluorescence in the brain tissue, but we have used it successfully for probe placement validation.

[BEFORE_START]
The mouse brain used for this protocol should be fixed in 4% PFA prior to the start of this protocol. See Mouse Cardiac Perfusion Fixation and Brain Collection V.5 for further details.

[STEPS]
SECTION: Day 1: Dehydrate the PFA-fixed mouse brain in a step gradient of Methanol
1. Prepare 10mL of the following Methanol concentrations: 

20% Methanol in Milli-Q Water
40% Methanol in Milli-Q Water
60% Methanol in Milli-Q Water
80% Methanol in Milli-Q Water


 

4. Remove the Methanol solution used in the previous step from the glass vial containing the mouse brain specimen and dispose of it in an appropriate waste container. Measure 100% Methanol into glass vial containing the mouse brain for 60 min. 
SECTION: Day 2: Delipidation with Dichloromethane
7. Remove 33% Methanol and 66% Dichloromethane solution from the glass scintillation vial using a glass serological pipet.
5. Repeat step 7 for a second 100% Methanol 60 min  wash.

6. Prepare 10mL of 33% Methanol and 66% Dichloromethane solution using 3.33mL Methanol and 6.66mL Dichloromethane. While a plastic serological pipet is adequate for dispensing Methanol, a glass serological pipet should be used to dispense Dichloromethane.


 

6.1. Leave the whole mouse brain in the 33% Methanol and 66% Dichloromethane solution   .
SECTION: Day 2: Delipidation with Dichloromethane
8. Using a glass serological pipet, measure 10mL of 100% Dichloromethane into the glass scintillation vial for a 20 min wash.

SECTION: Day 2: Delipidation with Dichloromethane
9. Repeat step 8 for a second 100% Dichloromethane 20 min wash.

2. Place whole mouse brain specimen (must be previously fixed in 4% PFA) in 20mL glass vial. Vial should be covered in foil to protect from light and remain covered throughout the rest of the protocol.
 

3. Dehydrate through H2O → Methanol series

Wash brain specimen with Methanol solution (diluted in water) for each step using the solutions prepared in step 1. Use a serological pipet to add and remove Methanol solution from the 20ml glass scintillation vial containing the brain specimen for each step.

20% Methanol for 60 min 
40% Methanol for 60 min
60% Methanol for 60 min
80% Methanol for 60 min

SECTION: Day 2: Delipidation with Dichloromethane
10. Brain should immediately be index matched with ethyl cinnamate following the second 100% Dichloromethane wash in step 9. See protocol Refractive Index Matching - Ethyl Cinnamate for instructions.
TITLE: Peptide C-Terminal Modification
AUTHORS: Cathy  Miller

[DESCRIPTION]
The post-translation modification of eukaryotic proteins by the addition of isoprenyl lipids at their C-termini was first observed in the 1970s and 1980s. Since then, more than a hundred proteins have been shown to be modified by C15 farnesyl or C20 geranylgeranyl groups, including most members of the Ras, Rho, and Rab families of G proteins. The C-terminal of the peptide is synthesized as an amide to neutralize the negative charge created by the C-terminal COOH. This modification is added to prevent enzyme degradation, to mimic native proteins, and in some cases to remove hydrogen bonding at the C-terminal of the peptides which may interfere with the assays.

Creative Peptides specialized in the custom synthesis of C-terminal modification peptides, providing a confidential and efficient service at competitive prices. Every step of peptide synthesis is subject to Creative Peptides' stringent quality control.
Typical delivery specifications include
HPLC chromatogram
Mass spec analysis
Synthesis report
Certificate of analyses

[STEPS]
TITLE: Immunohistochemistry
AUTHORS: Alexandra Nelson

[DESCRIPTION]
This protocol describes immunohistochemical staining of fixed brain sections.

[STEPS]
1. Sectioning
2. Blocking. 
Incubate sections in blocking buffer at Room temperature for 1-2 hours.
Blocking buffer is: 3% NDS (normal donkey serum)/ 0.1% triton (also called NDST) in PBS.
3. Primary Antibody. 
Prepare primary antibody in 3% NDS at desired concentration (make sure this is the final concentration in the well if you are adding more than one primary) and let sections sit on shaker   at 4 °C 

4. Wash. 
Wash sections 3-5 times in PBS for 10-15 minutes each at Room temperature  

5. Secondary Antibody. 
Prepare secondary antibody in 3% NDS at desired concentration and let sections sit on shaker for 2-4 hours at 4 °C  
 



6. Wash. 
Wash sections 3-5 times in PBS for 10-15 minutes each at Room temperature
7.  Mounting. 
Mount sections onto slides with Vectashield hardset mounting medium (with or without DAPI).
8. Imaging.
1.1. Using freezing microtome section brain at 30-35 μm
1.2. Place sections in PBS in a 24-well plate until ready for immunohistochemistry; if not performing immunohistochemistry that day, cover 24 well plate and place at 4ºC overnight
8.1. For low-magnification display of viral expression patterns or tyrosine hydroxylase staining, we typically use 10X stitched epifluorescence images.
8.2. For evaluation of individual neurons and overlap of fluorescent markers, we typically use 40X epifluorescence or confocal Z-stacks.
TITLE: Modified Leeming and Notman Agar
AUTHORS: Amy Gladfelter

[DESCRIPTION]
Media recipe for growth of Malassezia species.

[STEPS]
1. bacteriological peptone (Oxoid) 10 g

2. yeast extract 2 g

3. desiccated ox bile (Oxoid) 8 g

4. glucose 10 g

5. glycerol monostearate 0.5 g

6. agar 15 g

7. glycerol, 50% 20 mL

8. tween 60 5 mL

9. olive oil 20 mL

10. dH2O  955 mL

11. Sterilize by autoclaving.
TITLE: Intrapancreatic injection surgery
AUTHORS: Maria Jimenez Gonzalez, Rosemary Li, Sarah Stanley

[DESCRIPTION]
This is a protocol for performing intrapancreatic injection of Cholera Toxin Beta (CTb) in mice. 

[BEFORE_START]
-This study was approved by the Institutional Animal Care and Use Committee of Icahn School of Medicine at Mount Sinai
-Clean and sterilize properly all surgical tools needed.


[STEPS]
SECTION: Preparing surgery area
1. - Prepare the surgical space by placing the absorbant pad and the inhalatory anesthesia. Write down the mouse weight. 
- Anesthetize the animal by inducing anesthesia with 3% Isofluorane. Once anesthesia is induced, place animal in supine position and keep anesthesia at 1-1.5%.
- Apply eye lubricant (eg. Lacri Lube). Subsequently apply hair removal cream (eg. Nair) in the area of incision (left abdominal area). Apply Povidone/Yodine in the incision area. Apply Lidocaine afterwards. Apply Ethanol swab. Now the surgical area is ready for incision
SECTION: Surgery
2. -Make a vertical incision of 1 to1.5cm, left side of the abdomen. Gently cut first skin, then abdominal wall. Cut a piece of gauze and wet it in sterile PBS, place close to the incision point. With the help of cotton tips or arruga forceps, expose the pancreas and place it in the gauze. 
-  100 µg of CTb is dissolved in 5 µLSterile PBS. With the use of a 5 µL Hamilton syringe, perform 5 injections of  1 µL volume each . In the case of AAV injection, total volume of injection should range between 5 µL-20 µL, depending on the concentration needed.  Inject in several places of the pancreas (spread between head and tail of the pancreas). Every injection should be performed at a slow pace (5-10 seconds), waiting 10-15 seconds before removing syringe. 
- Place the pancreas back to the abdominal cavity. Suture (2-3 sutures) the abdominal wall, using Vicryl Suture 4-0, 1/2c RB1 (or similar). Close the skin with an autoclip applier. 






SECTION: Post-surgery care
3. After surgery, inject Buprenorphin (SC, 0.05-0.1 mg/kg) and place animal back to its cage. Analgesia should be injected the 2 following days, twice a day. Monitor animal weight and behavior. 
SECTION: CTb expression
4. Harvest tissues of interest: Coeliac ganglia, Nodose ganglia, Dorsal Root Ganglia 1 week after injection of CTb. In the case of AAVs, harvest tissues 3-4 weeks after injection.
TITLE: Protein A and Protein AG Sandwich ELISA
AUTHORS: Angel  Justiz-Vaillant

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This ELISA was used to study the interactions between Staphylococcal protein-A (SpA) and protein-AG (SpAG) with different immunoglobulin preparations from mammalian and avian species.</div></div>

[STEPS]
?. This ELISA was used to study the interactions between Staphylococcal protein-A (SpA) and protein-AG (SpAG) with different immunoglobulin preparations from mammalian and avian species. The 96 well microtiter plate was coated overnight at 4°C with 2 µg/µl per well of SpA in carbonate-bicarbonate buffer pH 9.6.
?. The plate was then treated with bovine serum albumin solution and washed 4X with PBS-Tween. 50 µl of immunoglobulins (1 mg/ml) is added and incubated for 1h at room temperature, and the microplate is rewashed 4X with PBS-Tween.
?. Then, 50 µL of peroxidase-labeled SpAG conjugate diluted 1:5000 in PBS-non-fat milk was added to each well and incubated for 1h at RT. The plate was washed 4X with PBS-Tween.
?. Then, 50 µL of 4 mg/mL o-phenylenediamine solution was added, and the plate was incubated for 15 min at RT in the dark. The reaction was stopped with 50 µL of a 3M H2SO4 solution.
?. The plate was visually assessed for color development and read on a microplate reader at 492 nm. A cut-off point was calculated as the mean of the optical density of the negative controls multiplied by two. The cut-off value was 0.34.
TITLE: Phenol-chloroform DNA purification
AUTHORS: Tomasz Suchan

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Because of the influence of salts on enzymatic reactions, Qiagen extractions can be purified before making RAD tags. First, it is necessary to perform a chloropane extraction (phenol / chloroform 50/50) by adding SDS to the aqueous phase (final conc. 0.1%), this eliminates contaminants introduced by the matrix of the column. Then, perform an ethanol precipitation followed by a wash with 70% ethanol. This is to eliminate the salt that was used to bind the nucleic acids to the column and also to remove EDTA (inhibitor!) which is 0.5 mM in the buffer AE. Eluting with water is nonsense as the pH is not optimal and thus yield is poor and the DNA is not buffered and there is always too much salt.</div></div>

[STEPS]
?. [Phenol-chloroform extraction]
To 100ul eluted DNA, add 0.5 ul of 20% SDS (*this may not be necessary as there are no cells) and 100 ul of phenol-chloroform.
?. [Phenol-chloroform extraction]
Vortex well.
?. [Phenol-chloroform extraction]
Centrifuge at room temperature for 5 min, at full speed (14,000 rpm).
?. [Phenol-chloroform extraction]
Pipette the aqueous phase (upper phase, aprox 80 ul, it is better to leave some DNA than to pipette phenol) to a new labeled tube.
?. [Phenol-chloroform extraction]
Discard original tube.
The tube contains phenol-chloroform.
?. [Phenol-chloroform extraction]
Proceed to ethanol precipitation.
?. [Ethanol precipitation]
Add 1/10 volume Na acetate 3M pH 4.8 or 5.2 (ie 8ul for 80 ul DNA solution)add 2 volumes ETOH 100% (storage -20°C) = 176 ulTotal volume: 264ul, possible with 264 ng of starting DNA - otherwise, you must add a carrier: tRNA, glycogen or linear acrylamide.
?. [Ethanol precipitation]
Vortex.
?. [Ethanol precipitation]
Put on dry ice for 30 min. or overnight at -20°C
?. [Ethanol precipitation]
Centrifuge at 4°C for 30 min at 14,000 rpm.
?. [Ethanol precipitation]
Discard the supernatant.
?. [Ethanol precipitation]
Wash with 500ul EtOH 70% (storage  4°C)
?. [Ethanol precipitation]
Centrifuge at 4°C for 5 min at 14,000 rpm.
?. [Ethanol precipitation]
Discard the supernatant
?. [Ethanol precipitation]
Quick spin
?. [Ethanol precipitation]
Pipette out the last drop of EtOH
?. [Ethanol precipitation]
Speed Vac for 3' or 5-7 min at room temperature.
?. [Ethanol precipitation]
Resuspend in 20ul of water ot Tris 10 mM pH 7.5 or 8.0
TITLE: Immunofluorescent Staining
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to stain cryosectioned mouse brain tissue.

[STEPS]
1. To cryo-sectioned brain tissue, wash with 1X phosphate buffered saline (PBS) for 3x 5-minute washes.
2. Incubate in blocking buffer for 1 hour at room temperature.
Blocking buffer: 10% serum + 0.5% Triton X-100 in 1X PBS
3. Wash tissue with 1X PBS.
4. Incubate in primary antibody diluted in blocking buffer overnight at 4oC.
5. Wash tissue with 1X PBS for 5x 5-minute washes.
6. Incubate in secondary antibody diluted in blocking buffer for 1 hour at room temperature.
7. Wash tissue with 1X PBS for 5x 5-minute washes.
8. If tissue wasn't previously mounted on a slide, mount on a superfrost plus slide and let dry at room temperature for at least 10 minutes.
9. Coverslip with fluorescent mounting medium and a #1.5 coverslip. Outline the coverslip with clear nailpolish.
TITLE: Terrific broth (TB) medium
AUTHORS: Andreas Sagen

[DESCRIPTION]
IBI’s Terrific Broth is used with Glycerol in cultivating recombinant strains of E. coli. Terrific broth is a highly enriched medium for improving yield in plasmid bearing E. coli. Recombinant strains have an extended growth phase in the medium. The addition of tryptone and yeast extract in the medium will allow higher plasmid yield per volume. Glycerol is used as a carbohydrate source in this formulation. Unlike glucose, glycerol is not fermented to acetic acid.

[BEFORE_START]
Prepare glassware by cleaning it, and ensure that scale is sufficiently calibrated

[GUIDELINES]
Follow step by step, unless stated otherwise. Equipment needed should be standard to a microbiology lab.

[STEPS]
SECTION: Terrific broth base solution
1. Fill the bottle with 200 mL distilled water

 

SECTION: Terrific broth base solution
2. Measure 11.8 g Yeast extract and 5.9 g Tryptone. Add 7.5 g for making agar plates

Materials:
 
 
 
SECTION: Terrific broth base solution
3. Add powdered solids into bottle, and use a magnetic mixer with a stir bar to mix for 5 min
SECTION: Terrific broth base solution
4. Add 100 mL Potassium phosphate buffer (1 M, 6.2 pH)
 

SECTION: Terrific broth base solution
5. Adjust pH while mixing to pH 7.2 using concentrated sodium hydroxide
SECTION: Terrific broth base solution
6. Add distilled water to a total of 400 mL
SECTION: Terrific broth base solution
7. Autoclave liquid at 121 °C for 15 min
SECTION: Terrific broth carbon solution
8. Fill the bottle with 300 mL double-distilled water
SECTION: Terrific broth carbon solution
9. Add 20 mL Glycerol and mix solution well

Materials:
 
SECTION: Terrific broth carbon solution
10. Adjust pH while mixing to pH 7.2 using concentrated sodium hydroxide
SECTION: Terrific broth carbon solution
11. Add distilled water to a total of 500 mL
SECTION: Terrific broth carbon solution
12. Autoclave liquid at 121 °C for 15 minAutoclave liquid at 121 °C for 15 min
SECTION: 500 mL Terrific broth
13. Add 100 mL carbon solution to base solution
SECTION: 500 mL Terrific broth
14. Aliquot medium in 100 mL units, and store refrigerated (4 °C)