eamag/protocols-with-steps huggingface dataset:
TITLE: X-ray Micro Computed Tomography (MicroCT) 
AUTHORS: Marta   Wawrzyniak, Nathalie  Weber, Simon  Blanchoud

[DESCRIPTION]
This protocol has been successfully used with colonial tunicates. It has been adapted to our needs based on the following publication: " MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues " by Brian D Metscher. 

MicroCT for comparative morphology: simple staining methods allow high-contrast 3D imaging of diverse non-mineralized animal tissues.

[STEPS]
SECTION: Fixation
1. Clean the slide with the colony of your interest. See Cleaning colonial ascidians V.2.

SECTION: Fixation
2. Anesthetize the animals following this protocol. See Whole colony fixation V.1


SECTION: Fixation
3. Fix the animals in 4% PFA   at Room temperature . See Whole colony fixation V.1

SECTION: Fixation
4. Wash twice in 3.3x PBS for 30 min .
SECTION: Fixation
5. Rinse tree times with 1x PBS and leave in 1x PBS for 60 min .


SECTION: Staining
6. Detach the colony with a razor blade and put in a 2mL tube containing  1 mL PTA /0.6 mL  IKI/ 0.4 mL  Ethanol. 

SECTION: Staining
7. Incubate for 5760 min  at Room temperature  on linear shaker (low speed), protect from light.


SECTION: Staining
8. Rinse quickly 3-4 times with 1x PBS.
SECTION: Mounting
9. Prepare 10 mL of 0.5 % volume low melting agarose.

SECTION: Mounting
10. Place the tip into a beaker filled with ice-cubes so that the agarose becomes solid. 
SECTION: Mounting
11. Cover the tip with fixing paste. 
SECTION: Mounting
12. Store at 4 °C or proceed to the next step. 

SECTION: Mounting
13. Mount the tip on the tomograph support using fixing paste in such way that it is perfectly straight.  
SECTION: Mounting
14. Place it inside the tomograph.
SECTION: Mounting
15.  Follow the tomograph's manual and proceed to scan.  
SECTION: Mounting
9.1. When the agarose has cooled down a bit, pipette 125-150 µL if using a 200ul tip or 750-800 µL  if using a 1mL tip (the tip size depends on the animal size).

SECTION: Mounting
9.2. Carefully place your colony vertically inside the tip (it should be as flat as possible, avoid folds).   
SECTION: Mounting
9.3. Pipette some more agarose in order to cover the animal entirely.  
SECTION: Mounting
16. After scanning, the tip with the sample can be stored at 4 °C for several weeks and eventually can be reused.
TITLE: Code_file_Caroline_Laborde_barrier_free_neighbourhoods_functional_independence_educational _inequalities 
AUTHORS: laborde_caroline

[DESCRIPTION]
Code file for 

"Do barrier-free neighbourhoods foster functional independence equally among older people with lower and higher educational status?"
by: Caroline Laborde, Joël Ankri and Emmanuelle Cambois 

last update: November 1st, 2022

[STEPS]
TITLE: Preparation and Immunohistochemistry of Mouse Small Intestine and Colon
AUTHORS: Nathan D Rossen

[DESCRIPTION]
Mice were euthanized, the small and large intestine collected, and fixed frozen sections prepared.  The microscope mounted tissue sections were stained using immunohistochemistry.  Expression of specific proteins and reporter proteins were visualized using microscopy.

[STEPS]
SECTION: Tissue Collection and Preparation
1. Mice 6 - 8 weeks old were euthanized by isolfurane administration followed by cervical dislocation.
SECTION: Tissue Collection and Preparation
2. Mice were transcardially perfused with 5ml ice cold PBS.
SECTION: Tissue Collection and Preparation
3. Whole small intestine and colon were collected and kept in PBS on ice.   The small intestine was dissected into three segments of approximately equal length.  
SECTION: Tissue Collection and Preparation
4. Each segment (proximal, medial, and distal small intestine and whole colon) was flushed with 5ml ice cold PBS, cut lengthwise, and laid flat with the mucosal surface facing up.  Each segment was further washed with ice cold PBS.
SECTION: Tissue Collection and Preparation
5. Tissue segments were washed with ice cold 4% PFA in PBS, and then laid flat in between two layers of filter paper and fixed in 4% PFA in PBS at 4C for either 4hrs or 24hrs.  

SECTION: Tissue Collection and Preparation
6. Tissue was rinsed in excess PBS and transferred to 30% sucrose solution for at least 16 hours.

SECTION: Tissue Collection and Preparation
7. Tissue was transferred to a mixture of 50% OCT, 15% sucrose in PBS and rolled on a wooden stick.  
SECTION: Immunohistochemistry
9. Slide-mounted tissue sections were thawed and air dried for 30 minutes
10. Sections were washed twice for 10 minutes in PBS and once for 10 minutes in .2% Triton X-100 in PBS.
SECTION: Tissue Collection and Preparation
8. Tissue was frozen in OCT as a "swiss roll" and sectioned into 5um or 20um sections on a cryostat and kept at -80C.
11. Sections were blocked for 1 hour in blocking buffer at room temperature.
12. Sections were incubated with primary antibodies in blocking buffer overnight at 4C.
13. Sections were washed thrice for 10 minutes in .2% Triton X-100 in PBS.
14. Sections were incubated with secondary antibodies and DAPI in blocking buffer for 1-2 hours at room temperature.
15. Sections were washed twice for 10 minutes in .2% Triton X-100 in PBS and twice for 10 minutes in PBS.
16. Sections were mounted in Prolong Gold Mounting Media.
TITLE: U Cinn - NEFA Concentration
AUTHORS: Patrick Tso, Dana Lee

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:  </span></div><div class = "text-block">Quantitative determinations of non-esterified fatty acids in plasma/serum/lymph will be made using the NEFA-HR enzymatic colorimetric method assay.</div></div>

[STEPS]
?. Prepare working Color Reagent Solutions A and B. A. Reconstitute Color Reagent A with a portion of Solvent A and then transfer entire contents into Solvent A bottle, rinsing Color Reagent vial several times. B. Reconstitute Color Reagent B with a portion of Solvent B and then transfer entire contents into Solvent B bottle, rinsing Color Reagent vial several times.
?. Locate working Standard (1mmol/L or 1 mEq/L).   THIS ASSAY DOES NOT REQUIRE A SERIAL DILUTION
?. Using a 96 well flat bottom plate, into separate wells, pipette 5μL of deionized water, 1mMstandard, or sample to be assayed.
?. Add 200μL of Color Reagent Solution A to all wells.
?. Mix well and Incubate plate for 5 minutes at 37°C.
?. Measure the absorbance of each well at 550nm (sub:660nm).  This measurement (Abs1) will serve as the sample blank.
?. Add 100μL of Color Reagent Solution B to all wells.
?. Mix well and Incubate plate for 5 minutes at 37°C.
?. Measure the absorbance of each well at 550nm (sub:660nm).  This will be your Abs2 value.
?. Obtain the final absorbance (Sampleabs) by subtracting the first reading (step 5) from the second reading (step 8). *
?. Plot the absorbance vs. concentration to construct the calibration curve.  A linear calculation model should be used.
?. To calculate sample concentration by calculation use the following formula: Sample Conc. = (Sample Absorbance/Standard Absorbance) * Standard Concentration *The sample blank absorbance (Abs1) from the first measurement (step 5) should be multiplied by a Factor (F) in order to correct for changes in volume, as follows:  F=(Sample vol + R1 vol) / (Sample vol. + R1 vol + R2 vol)  For this assay:  F = (5+200) / (5+200+100)  =  0.67  Therefore:   Sampleabs  = Abs2 – (Abs1 * 0.67) Specimen:  Serum or Plasma.  Specimen stable for 7 days at 2-8°C or 3 months at -20°C. Assay Linearity: 4.0 mEq/L Reagent Stability:  7 days at 2-8°C Stability of Final Reaction:  60 minutes
TITLE: In vivo CAR T cell tumor control assay 
AUTHORS: Andrea R Daniel

[DESCRIPTION]
This protocol describes methods for a HCC1954 cell implanted orthotopic tumor model in mice with delivery of T cells expressing HER2 CAR. Tumor volumes are measured to evaluate tumor control by CAR T cell therapy.

[STEPS]
SECTION: HCC1954 orthotopic tumor model
1. Six- to 8-week-old female immunodeficient NOD/SCID gamma (NSG) mice were obtained from Jackson Laboratory and then housed in 12-h light/dark cycles, at an ambient temperature (21 ± 3 °C) with relative humidity (50 ± 20%) and handled in pathogen-free conditions
SECTION: HCC1954 orthotopic tumor model
2. HCC1954s were maintained in DMEM/F12 supplemented with 10% FBS, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin.
SECTION: HCC1954 orthotopic tumor model
3. Resuspend cells in 2ml PBS (5x10^7 cells/ml), then diluted 1:1 with Matrigel (2.5e7/ml)
thus 2.5e6 per 100ul.

SECTION: HCC1954 orthotopic tumor model
4. 2.5x 106 HCC1954 cells are implanted orthotopically into the mammary fat pad of NSG mice in 100μL volume of 50:50 (v:v) PBS:Matrigel.

SECTION: HCC1954 orthotopic tumor model
5. Upon detection tumor volumes were calculated based on caliper measurements using the formula volume = ½ (Length × Width2). Measure tumors every 4-6 days.

SECTION: CAR T cell delivery
6. Human donor T cells were expanded for 9-11 days post-transduction with HER2-CAR-2A-GFP
and HER2-CAR-2A-BATF3 constructs before delivery to tumor bearing mice. 

SECTION: CAR T cell delivery
7. Transduction rates were measured on the day of treatment using flow cytometry and transduction rates exceed 80%.

SECTION: CAR T cell delivery
8. T cells were resuspended at 50 x 106 CAR+ cells mL-1 in 1X PBS and serially diluted to the appropriate cell concentrations for 200 μL injections of either 10 x 106, 2 x 106, 5 x 105,
2.5 x 105, or 1 x 105 HER2 CAR+ T cells. 

SECTION: CAR T cell delivery
9. 21 days after tumor implantation  randomize mice into groups for CAR T cell injections. Deliver CAR T cells intravenously by tail vein injection.

SECTION: CAR T cell delivery
10. Measure the length and width of tumors every 4-6 days using calipers.
SECTION: CAR T cell delivery
11. Mice are euthanized before reaching a tumor volume of 2,000 mm3, the upper threshold defined by the Duke Institutional Animal Care and Use Committee.
TITLE: Zymoclean™ Gel DNA Recovery
AUTHORS: Sze-Xian Lim

[STEPS]
?. Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it into a 1.5 ml microcentrifuge tube.
?. Weight empty 1.5 ml microcentrifuge tube.
?. Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. add of ADB to every of agarose gel).
[ADB]
[agarose gel]
?. Incubate at to for to until the gel slice is completely dissolved.
[minimum]
[maximum]
[minimum]
[maximum]
?. Transfer the melted agarose solution to a Zymo-Spin™ Column in a Collection Tube.
?. Centrifuge for to seconds.
[minimum]
[maximum]
?. Discard the flow-through.
?. Weight tubes with agarose gel and subtract the weight of the empty tube.
?. Add of DNA Wash Buffer to the column and centrifuge for  .
[DNA Wash Buffer]
?. Discard the flow-through.
?. Repeat the wash step by adding of DNA Wash Buffer to the column and centrifuge for  .
[DNA Wash Buffer]
?. Add at least  of DNA Elution Buffer directly to the column matrix.
[DNA Elution Buffer]
?. Place column into a 1.5 ml tube and centrifuge for to seconds to elute DNA.
[minimum]
[maximum]
TITLE: Prototype Protocol
AUTHORS: George Moberly

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Prototype Protocol</div></div>

[STEPS]
?. Add 200 μl SWS to the tube. Pipette to mix.
?. Place on the 50°C heat block for 30 minutes.
?. Immediately place on a magnetic stand and wait until the liquid is clear (~2 minutes).
?. Remove and discard all supernatant from the tube.
?. Remove from the magnetic stand. 1. Add 200 μl SWS to the tube. Pipette to mix.
TITLE: Cloning by Gibson Assembly 
AUTHORS: Eric ECS Cordeiro-Spinetti

[DESCRIPTION]
Gibson assembly is a molecular cloning method that allows for the joining of multiple DNA fragments in a single, isothermal reaction. It is named after its creator, Daniel G. Gibson, who is the chief technology officer and co-founder of the synthetic biology company, Telesis Bio. - Wikipedia


Daniel G. Gibson, of the J. Craig Venter Institute, described a robust exonuclease-based method to assemble DNA seamlessly and in the correct order, eponymously known as Gibson Assembly. The reaction is carried out under isothermal conditions using three enzymatic activities: a 5’ exonuclease generates long overhangs, a polymerase fills in the gaps of the annealed single strand regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps. This method has been widely adopted and is a major workhorse of synthetic biology projects worldwide.

[STEPS]
1. PCR (vector and insert)
Tm vector primers =  oC
Tm insert primers =   oC 
2. Clean-up PCR products with 1 µL Dpn1 for 30 min at 37 °C

3. Purify PCR products and resuspend in lowest volume possible (5-10 uL)
4. Set up Gibson ligation
    Vector = 50-100ng
    Molar ratio Vector/Insert = 1:1-3
5. Add to Gibson Master Mix
6. Incubate for 60 minat 50 °C

7. Transfer 1-2 µL into 50µL suspension of E.coli
8. Incubate on ice for 30 min

13. Select colonies for sequencing
9. Heat shock at40 °C for 30 seconds

10. Transfer to 300 µL of outgrowth media 

11. Incubate in shaker for 60 min at 37 °C

12. Plate on antibiotic containing plate and grow 60 min
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
This is a 0.05M Tris-Cl (pH 8.0), 0.01M EDTA resuspension buffer containing RNase A

[GUIDELINES]
Storage condition - 4°C after adding RNase A.<br /><strong>Note</strong>: Buffer compositions are given per liter of solution. Buffer calculations are based on Tris Base adjusted to pH 8.0 with HCl (Tris-Cl).  Not recommended to be made with Tris-HCl due to acidity of solution (EDTA-Na<sub>2</sub>-2H<sub>2</sub>O needs a pH above 8.0 to dissolve), but if Tris-HCl is used, amount should be adjusted for correct molarity (0.05M) and NaOH should be used to bring pH up to 8.0.  This buffer may be autoclaved prior to addition of RNase A or it may be 0.2 μm filter-sterilized.

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: Verhoeff’s Iron Hematoxylin Staining
AUTHORS: Jeff Z. Chen, Deborah A. Howatt, Michael K. Franklin, Jessica J. Moorleghen, Hisashi Sawada, Hong S. Lu, Alan Daugherty

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol is used to stain elastic tissue.</div><div class = "text-block">This results in elastic fibers and nuclei stained black.</div><div class = "text-block">Staining of elasin allows for quantification of elastin breaks.</div></div>

[STEPS]
?. [Elastin Staining]
Acetone to fix
If slides keep falling off slide, air dry for 30 mintues before fixation
?. [Elastin Staining]
Water (wash)
?. [Elastin Staining]
Verhoeff’s iron hematoxylin
?. [Elastin Staining]
Water (3 washes)
?. [Elastin Staining]
Ferric chloride (2% wt/vol)Water 3 washesCheck slide for differention at 1m30s and for every 30s afterward
* Halt staining when nuclei and elastin fibers are still black with weak background.*** If over-differentiated, go back to the step 3.
?. [Elastin Staining]
Ethanol (95% vol/vol)
?. [Elastin Staining]
Ethanol (100% vol/vol)
?. [Elastin Staining]
Xylene
?. [Elastin Staining]
Air dry uncovered
?. [Elastin Staining]
Permount and coverslip
TITLE: Yale Murine TMC - Tissue Preparation Protocol 
AUTHORS: Yun-Hee Youm, Yufeng Liu, Vishwa Deep Dixit, Rong Fan, Bo Tao

[DESCRIPTION]
Protocol for embedding murine spleen, thymus and lymph node.

[STEPS]
SECTION: RNA Buffer (use before embedding tissue in OCT)
1. 1x PBS-MC Recipe
All prepared in DNAse RNAase EDTA free deionized water
MgCl2 (1 mM, add 1 ml of 1 M MgCl2 stock solution into 1 L of 1x PBS)
CaCl2 (0.1 mM, add 2 ml of 0.1 M of CaCl2 stock soution into 1 L of 1x PBS)
4% paraformaldehyde (PFA), optional
1 X Protease inhibitors (Catalog # 04693132001, Roche-cOmplete , EDTA-free Protease Inhibitor Cocktail, Tablets provided in EASYpacks)
1X Phosphatase inhibitor (Catalog # 4906837001; PHOSS-RO; Roche-PhosSTOP™ ; sufficient for 20-10 ml buffer preparations, suitable for tissue processing and immunoprecipitation)
RNAse inhibitors (Invitrogen SUPERase•In RNAse Inhibitor (20 U/μL ; Catalog # AM2696)

SECTION: RNA Buffer (use before embedding tissue in OCT)
2. Filter sterilize with 0.22 μm syringe filter.  Store at 4°C before use.  Use within 2 weeks.
SECTION: RNA Buffer (use before embedding tissue in OCT)
3. Incubate tissue on ice in the buffer for 15 minutes or during the collection.
SECTION: Tissue Preparation/Embedding
4. Collect tissue in PBS on ice, wash if necessary
SECTION: Tissue Preparation/Embedding
5. Dehydration/Fixation
SECTION: Tissue Preparation/Embedding
5.1. Incubate the tissue in 20% sucrose/PBS solution or 4% paraformaldehyde and allow it to sink to the bottom of the vial at overnight at 4°C.
SECTION: Tissue Preparation/Embedding
6. Embedding
SECTION: Tissue Preparation/Embedding
6.1. Place the fresh tissue in the center of the mold (Tissue-Tek Cryomold #4566) filled with OCT (Tissue Plus OCT Compound, Fisher Scientific catalog # 23-730-571).  Be careful to remove any air bubbles.  Remove air bubbles in OCT bottle, then squeeze out.
SECTION: Tissue Preparation/Embedding
6.2. Orient the OCT-embedded tissue into the desired position in the mold.  Carefully squeeze out more OCT on top of the tissue, again avoiding bubbles.  Squeeze enough OCT out until none of the tissue remains exposed. 
SECTION: Tissue Preparation/Embedding
6.3. Freeze the tissue block on dry ice (or in cryostat).
SECTION: Tissue Preparation/Embedding
6.4. Wrap each block in aluminum foil or place into a small plastic bag.  Store the tissue block at -80° C until ready for sectioning.
TITLE: NCEM Drop - Inactivation of sample in solution (TM-014)
AUTHORS: sandra.crameri

[DESCRIPTION]
This inactivation procedure is used to inactivate a sample for Biological Transfer out of PC4, proir to Conventional NCEM.

[STEPS]
SECTION: Inactivation
1. Add 50 µL 16 % volume   to a Sarstedt tube with o-ring (STK#:2056-U, Catalog #61226C2).

SECTION: Inactivation
2. Add 150 µL sample to the   already in the tube. Leave contact time 10 min 


SECTION: Inactivation
3. Adsorb 10 µL sample to grid 10 min , inspect to ensure sample doesn't dry out.

SECTION: Inactivation
4. Drain excess sample from grid using fliter paper, leave wet.
SECTION: Inactivation
5. Stain   1 min 

SECTION: Inactivation
6. Drain & dry using filter paper
TITLE: CPMU
AUTHORS: Bjorn Bartholdy, a.g.henry

[DESCRIPTION]
Mineralising solution for the oral biofilm. From Sissons et al. 1991.

[BEFORE_START]
This solution MUST be prepared in a sterile and starch-free environment.

[STEPS]
1. Add 300 mL distilled (or deionized) dH2O to a 1000 mL  beaker, with stirring and heat 60 °C 
2. Add:

- 1.55 g   
- 1.44 g   
- 0.72 g   
- 0.08 g   
- 30 g   

3. Add the remaining 700 mL  and keep stirring until precipitate has completely dissolved
Store in fridge at 4 °C
TITLE: OMS Atlas OCT Spatial Mapping - Limited
AUTHORS: Brett Johnson, Danielle Galipeau, George Thomas

[DESCRIPTION]
This protocol describes the procedure by which the OMS Atlas serially sections an OCT block, prepares the resulting slides and samples, and then distributes the specimens for downstream analysis.

[BEFORE_START]
Transfer OCT blocks to OHSU Knight Histopathology Shared Resource (HSR) for sectioning and processing.

[STEPS]
SECTION: Preparation
1. Verify the identity of the OCT block to be cut against written request for sectioning.
SECTION: Preparation
2. Remove OCT block from -80 °C freezer and acclimate to cryostat (-20 °C ) for minimum of 180 min.

SECTION: Preparation
3. Label all slides and cryotubes with a unique BEMS ID and Part#, corresponding to the written request and OCT spatial map (below).
 

SECTION: Sectioning
4. Affix OCT block to cryostat chuck.
SECTION: Sectioning
5. Orient and face block to get adequate amount of core.
Note: Avoid excessive facing to reduce tissue loss.
SECTION: Sectioning
6. Set cryostat to 5 micron sections.
Note: All sections cut from here on should be sequential. The serial order, adjacency, and consistent orientation of the sections are all important factors. Please note any deviations from the protocol.
SECTION: Sectioning
7. Cut three sections at 5 microns (Part#1-3) and affix onto appropriately labeled slide according to OCT spatial map (step #3 above).
SECTION: Sectioning
8. Place all slides and remaining OCT block in -80 °C freezer.
Note: No slides are to be fixed under this protocol.

SECTION: Processing
9. Perform hematoxylin and eosin (H&E) staining on slide labeled Part#1 (see OCT spatial map in step #3 above).
SECTION: Processing
10. Deliver unstained slides (Part#2 and 3) and remainder OCT block (Part#4) to BioLibrary for distribution.
Note: Keep samples frozen at all times. Store at -80 °C. Transfer/ship on dry ice.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>PD-L1 LDT on Dako Autostainer Link 48 (ASL48) using the concentrate 22C3 antibody. This protocol was developed in the laboratory of Dr. Paul Hofman and Dr Marius Ilie (Pasteur Hospital, Nice, France).</p>
<p>For the purpose of protocol development 95% of clinical specimens consisted of surgical resection specimens and the remaining 5% were bronchial biopsies and ransbronchial mediastinal lymph node biopsies.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Ez Burn Keto Gummies Canada Reviews! Shocking Benefits! Scam Or Legit!
AUTHORS: Ez Burn Keto Gummies  Canada

[DESCRIPTION]
What are EZ Burn Keto Gummies?

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[STEPS]
TITLE: Limited storage of spittlebugs or green leafhoppers (under one week)
AUTHORS: Niels Appelman

[STEPS]
?. Cover the bottom of a PET container (e.g. https://www.world-of-bottles.co.uk/Glass-bottles/250ml-white-PET-jar-Bella-Mia-white.html) with moisturised single use towels (wetten with tapwater and squeeze to remove excess water) and provide airholes in container with an (injection) needle.
?. Fill container with suitable plants
?. Invert the tube in which insects are temporarily stored and allow insects to climb up
?. While holding the tube above the new storage container, uncrew its cap and forcefully tap the back of the tube such that the insects drop into their new storage container.
?. Swiftly close the new storage container.Note: this step must be performed as quickly as possible, as it is crucial to minimize the time insects get to recover and escape.
?. Move insects into experimental setups as soon as possible, preferably within 2 days. Survival rate is highly variable. Upto 7 days of survival was achieved.