eamag/protocols-with-steps huggingface dataset:
TITLE: UC Davis - Immunohistochemistry Factor VIII
AUTHORS: Jennifer Rutkowsky

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary: </span></div><div class = "text-block">Factor VIII is a glycoprotein present in human plasma, human endothelial cells, megakaryocytes and platelets. Immunohistochemical staining for factor VIII related antigen could be used to determine if the benign and malignant neoplastic lesions are of endothelial origin. Furthermore, it can be utilized to determine vessel density in a tissue of interest</div><div class = "text-block"><span style = "font-weight:bold;">Modified from</span><span>: IHC Methods and Materials VMTH - Anatomic Pathology, UC-Davis. </span></div></div>

[STEPS]
?. Immunohistochemistry was performed on four-micron thick, formalin-fixed, paraffin-embedded tissue sections, mounted on charged slides, and air-dried overnight at 37º C.
?. Sections were deparaffinized through xylene to 100% reagent alcohol, and then treated with 0.3% hydrogen peroxide in 100% methanol for 30 minutes.
?. Sections were rehydrated to deionized water through 95% and 70% reagent alcohols. Antigen retrieval was performed on sections for IBA-1 with heat induced epitope retrieval in a Black & Decker Steamer using Target Retrieval Solution, pH 6 (Dako S1699) for 30 minutes at 95ºC, followed by a 20 minute cool down.
?. Retrieval was performed on sections for Factor VIII with Proteinase K (Dako S3020) at room temperature for 10 minutes.
?. After antigen retrieval, slides were rinsed in deionized water and placed in 0.1M Phosphate Buffered Saline, pH 7.4 (PBS). The antibody diluent and blocking reagent were PBS-Tween 20 (0.02%) and 10% normal horse serum (NHS) in PBS-Tween 20, respectively.
?. Sections were blocked for 20 minutes with antibody diluent and primary antibodies were applied without rinsing and incubated for 1 hour.    a. All post-antigen retrieval incubations are in a humidity chamber at room temperature.
?. After primary incubation, samples are rinsed twice for three minutes with PBS-Tween 20 between each subsequent reagent application.
?. A single step, polymer based HRP (BioCare Medical, RC542H) was applied for 30 minutes to label rabbit anti-IBA-1. A dual step, biotin-avidin based HRP (Biocare Medical, 4+ Detection System GR608) was applied for 10 minutes to link rabbit anti-Factor VIII.
?. Streptavidin-HRP (Biocare Medical HP604) was applied for 10 minutes to label the biotin link.
?. All labels were visualized with NovaRed for peroxidase (Vector SK-4800), per manufacturer’s instructions.
?. Sections are counterstained in Mayer’s Hematoxylin, air dried and coverslipped.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Please contact Dr. Steven Wilhelm (wilhelm@utk.edu) for additional information regarding this protocol.</p>
<p> </p>
<p>This cleaning protocol is meant to be used when there is an excess of particulate buildup in the FCM. Blockages can be detected by the failure of the FCM to successfully plot data in the scatter plot even though the machine is in the ‘acquire sample’ step. This will be visualized on the scatter plot as either extreme alterations to the forward and side scatter plot (a Etch A Sketch like drawing) or a complete absence of cell populations that should be there otherwise. If blockages are not addressed immediately, counts will be significantly affected because the machine cannot tell when there is a blockage- it will think volume is passing through the flow cell when it isn’t, which will artificially lower your cell counts.</p>

[BEFORE_START]
<p>It is extremely important to check the ICF vial for particulates and replace ICF if necessary.</p>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.
TITLE: Immunofluorescence Staining
AUTHORS: Toby J Curless, Hemanth Ramesh Nelvagal, Zane Jaunmuktane

[DESCRIPTION]
The protocol describes immunofluorescence staining on brain sections.

[STEPS]
SECTION: Preparation
1. Generate tissue sections using standard microtome sectioning protocols. 
SECTION: Preparation
2. Heat tissue dry tissue sections for 60 min at 60 °C
SECTION: De-paraffinisation and Immunofluorescence
3. 
 



De-paraffinise sections by 5 min min washes in xylene (x3), 5 min 100 % ethanol (x1), 5 min  70 % ethanol (x1) and 5 min DEPC H2O (x1).

SECTION: De-paraffinisation and Immunofluorescence
4. Rinse sections in PBS.
SECTION: De-paraffinisation and Immunofluorescence
5. Encircle tissue with a hydrophobic ImmEdgeTM PAP pen (Vector laboratories) to contain solution.
SECTION: De-paraffinisation and Immunofluorescence
6. Incubate tissue with H2O2for 15 min mins at room temperature.

SECTION: De-paraffinisation and Immunofluorescence
7. Wash tissue 3x in PBS.
SECTION: De-paraffinisation and Immunofluorescence
8. Antigen retrieval – heat 1 container (250 ml) DEPC H2O, and 1 container (250 ml) CC1 buffer to
99 °C using a steamer (BRAUN tribute collection).

SECTION: De-paraffinisation and Immunofluorescence
9. Add slides to heated DEPC H2O for 10 s, then transfer to CC1 buffer and incubate at 99 °C for 15 min.


SECTION: De-paraffinisation and Immunofluorescence
10. Remove slides and was 3x with PBS for 5 min mins total.

SECTION: De-paraffinisation and Immunofluorescence
11. Block sections with 10 % normal goat serum in PBS for 120 min.

SECTION: De-paraffinisation and Immunofluorescence
12. Wash tissue in PBS.
SECTION: De-paraffinisation and Immunofluorescence
13. Incubate sections in primary antibodies (antibodies diluted in PBS) at 4 °C 120 min.


SECTION: De-paraffinisation and Immunofluorescence
14. Following overnight incubation, wash the sections in PBS 3x for a total of15 min and shake off excess PBS after each 5 min interval.

SECTION: De-paraffinisation and Immunofluorescence
15. Incubate sections with 1:100 secondary antibodies (e.g., Alexa Fluor 488-conjugated anti-mouse secondary antibody (Invitrogen) and Alexa Fluor 568-conjugated anti-rabbit secondary antibody (Invitrogen)) (for 30 min mins at room temperature in the dark.

SECTION: De-paraffinisation and Immunofluorescence
16.  Wash sections in PBS 3x for a total of 15 min – whilst incubating, remove Antifade mountant (P36980, Thermo Fisher Scientific) from refrigerator and bring to room temperature.

SECTION: De-paraffinisation and Immunofluorescence
17. Counterstain sections with Hoechst (1:15000 (diluted in DEPC H2O)) for 30 s sec

SECTION: De-paraffinisation and Immunofluorescence
18. 4 °C Rinse sections in PBS 2x for a total of 6 min

SECTION: De-paraffinisation and Immunofluorescence
19. Coverslip slides using Antifade mountant (P36980, Thermo Fisher Scientific) and store slides in the dark at 4 °C.

SECTION: De-paraffinisation and Immunofluorescence
20. Capture slides on an epifluorescence microscope (Leica) using Alexa 488 filters.
TITLE: Database-driven protein solubilization
AUTHORS: Caroline Brown, Snehasish Ghosh, Kallol Gupta

[DESCRIPTION]
This is a protocol for using the extraction database found at www.polymerscreen.yale.edu for optimal extraction of membrane proteins into native nanodiscs.

[STEPS]
1. Choose your protein of interest.
2. Visit www.polymerscreen.yale.edu to search for your protein in the extraction database. This will provide you with the polymer to use for optimal protein extraction.
3. As an example, below are representative organellar markers and the optimal polymer for extraction:
TMEM192 --> AASTY650
TGN46 --> ChloroSMA60
OMP25 --> SMA200
VAPA --> AASTY650
KRas --> ChloroSMA80
4. Once the optimal polymer has been determined, express the protein of interest, or harvest cells for endogenous solubilization.
5. Resuspend cellular membranes in the chosen polymer and incubate at 4 °C with rotation for 120 min  hours.

6. Ultracentrifuge samples ay 200,000xg for 60 min  hours to remove insoluble material.
TITLE: Golden Gate Assembly (Protocol for NEB® Golden Gate Assembly Mix)
AUTHORS: Alba Balletbó

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Golden Gate Assembly and its derivative methods exploit the ability of Type IIS restriction endonucleases (REases) to cleave DNA outside of the recognition sequence. The inserts and cloning vectors are designed to place the Type IIS recognition site distal to the cleavage site, such that the Type IIS REase can remove the recognition sequence from the assembly.</div></div>

[STEPS]
?. [Assembly Reactions]
Set up 25 µl assembly reactions as follows:  ABC1Assembly ReactionNegative Control (If desired)2pGGA Destination Plasmid*, 75 ng/μl1 μl (75 ng)1 μl (75 ng)324 precloned inserts cloned into pMiniT 2.0, 100 ng/ul each plasmid0.75 µl (75 ng) each, (18 µl total)-4T4 DNA Ligase Buffer (10X)2.5 μl2.5 μl5T4 DNA Ligase, 2000 U/µl0.5 μl (1000 units)0.5 μl (1000 units)6BsaI-HFv2, 20 U/µl1.5 μl (30 units)1.5 μl (30 units)7Nuclease-free H2O1.5 µl19.5 μl
ABC1Assembly ReactionNegative Control (If desired)2pGGA Destination Plasmid*, 75 ng/μl1 μl (75 ng)1 μl (75 ng)324 precloned inserts cloned into pMiniT 2.0, 100 ng/ul each plasmid0.75 µl (75 ng) each, (18 µl total)-4T4 DNA Ligase Buffer (10X)2.5 μl2.5 μl5T4 DNA Ligase, 2000 U/µl0.5 μl (1000 units)0.5 μl (1000 units)6BsaI-HFv2, 20 U/µl1.5 μl (30 units)1.5 μl (30 units)7Nuclease-free H2O1.5 µl19.5 μl
?. [Assembly Reactions]
Mix gently by pipetting up and down 4 times.
?. [Assembly Reactions]
Briefly microcentrifuge (1 second) to bring material to the bottom of tube.
?. [Assembly Reactions]
Transfer to thermocycler and program as follows: (5 min 37ºC → 5 min 16ºC) x 30 cycles followed by 5 minutes 60ºC. If reactions are done overnight, add a 4ºC terminal hold to the protocol, but repeat the final 5 minutes 60ºC step the next day before the transformations.
?. [Transformation]
For each assembly, thaw a 50 µL tube of NEB 10-beta competent E. coli cells on ice for 5–10 minutes.
?. [Transformation]
Add 2 µL of the assembly reaction; gently mix by flicking the tube 4-5 times.
?. [Transformation]
Incubate on ice for 30 minutes.
?. [Transformation]
Heat shock at 42ºC for 30 seconds.
?. [Transformation]
Place back on ice for 5 minutes.
?. [Transformation]
Add 950 µL of room temperature NEB 10-beta/Stable Outgrowth Medium. Incubate at 37ºC for 60 minutes, shaking vigorously (250 rpm) or using a rotation device.
?. [Plating]
Warm LB agar plates containing chloramphenicol (for pGGA) at 37ºC for 15 minutes.
?. [Plating]
Mix the cells thoroughly by flicking the tube and inverting, then spread 100 µL outgrowth onto each plate.
?. [Plating]
Incubate the plates overnight at 37ºC, or 24 hours at 30ºC, or 48 hours at 25ºC.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>This was a past assay that we no longer in use. For our favoured Measles TaqMan test, please refer to the <em>MeV N TaqMan</em> protocol.</p>
<p>The protocol aims to amplify measles virus (MeV) strains and not other viruses.</p>
<p>Michael Lyon designed the original version of this unpublished in-house assay in 1999. It was superseded in 2003 but occassionally brought out as needed for troubelshooting. The superseding test was a first generation MGB-TaqMan protocol called <em>Measles MGB </em>TaqMan <em>FAST.</em></p>
<p>The assay targets the fusion (F) gene region and is designed as a qualitative test for investigating MeV infection of humans.</p>

[BEFORE_START]
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<div>
<ul>
<li>If using a different brand or model of real-time thermocycler, check the concentration of ROX is adequate.</li>
<li>Method assumes the user is familiar with the thermocycler and software used to run the protocol and with PCR in general.</li>
</ul>
</div>
</div>
</div>
</div>
</div>
</div>
</div>
</div>

[STEPS]
?. 
?. 
?. 
?. 
?.
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Transmission of bacteria from inanimate surfaces in healthcare associated environments is an important source of hospital acquired infections. A number of commercially available medical devices promise to fulfill antibacterial activity to reduce environmental contamination. Under usual ambient air conditions of hospital rooms no condensation of humidity is expected on inanimate surfaces. In contrast, current standardized methods for the analysis of antibacterial activity of solid surfaces in general use mostly planktonic bacterial cells which are kept in thin liquid or agarose layers on tested surfaces. Therefore, we developed a touch transfer assay modeling fingerprint transmission to investigate the antibacterial activity of surfaces in a dry state. We suggest the newly developed touch transfer assay as a new additional tool for the assessment of potential antimicrobial surfaces prior utilization in hospital environments.</p>

[STEPS]
?. 
?. 
?. 
?.
TITLE: Assembly of plasmids for LacI genotype-phenotype landscape measurement
AUTHORS:  Drew S Tack, Nina Alperovich,  Olga Vasilyeva, David Ross

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Here, we describe protocols for assembly of two different plasmids used for the measurement of the genotype-phenotype landscape of the LacI protein in </span><span style = "font-style:italic;">E. coli</span><span>: a Library Plasmid (pTY1, Figure 1a) used for the landscape measurement of the sensor library, and a Verification Plasmid (pVER, Figure 1b) used to verify the function of approximately 120 sensors from the library chosen to test the accuracy of the landscape measurement and to demonstrate the range of sensor function. We also describe the protocol used for the creation of the LacI sensor library in </span><span style = "font-style:italic;">E. coli</span><span style = "font-style:italic;">.</span></div></div>

[STEPS]
TITLE: Surgical Removal of Sea Cucumber Gonads
AUTHORS: Jon Eilers

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is a protocol for sea cucumber gonad removal without killing the sea cucumber or causing evisceration. </div></div>

[STEPS]
?. Anesthetize Sea Cucumber as described in the Sea Cucumber Anesthetizing protocol: dx.doi.org/10.17504/protocols.io.5nyg5fw
?. Locate the gonadopore just posterior to the head of the sea cucumber and pinch the skin just behind it
?. Using the pointed side of the dissection scissors, push into the body wall of the sea cucumber approximately one centimeter posterior to the gonadopore. When it is no longer difficult to push the scissor blade in, that means you have pierced the body wall. Now angle the scissors towards the back of the sea cucumber and cut a one to two centimeter incision.
?. Remove the scissors once the cut has been made. The body cavity pressure should push some fluid and the gonads out of the incision.
If the cut is made too far back on the body,  the respiratory tree and intestines are likely to also be expelled through the incision.
?. Remove the gonads and put into falcon tubes on ice. Push any internal organs back into the sea cucumber using the spatula.
If too much of the intestines or respiratory tree are expelled through the incision and then pushed back into the body cavity, the sea cucumber will likely eviscerate once the anesthetic wears off
?. Using suture, forceps, and hemostat forcep, begin stitching the wound closed using standard medical stitching techniques. Tutorials can be found on youtube: https://www.youtube.com/watch?v=FrCv-odr_QE
?. Once the wound is stitched closed, place the sea cucumber back in its container
The wound should completely heal in 2-3 days. Once healed, cut the stitches and remove them. If the stitches are left in too long a lesion will form.
TITLE: LUHMES (lund human mesencephalic) culturing and differentiation protocol
AUTHORS: Mallory Wright, William J Buchser, ckremitz, jwaligor, bachman@wustl.edu

[STEPS]
SECTION: LUHMES coating protocol
1.  
0.1 mg/mL
50 µg/µL
Thaw an aliquot of Poly-L-Ornithine solution at room temperature.
SECTION: LUHMES Passaging (~ every 2-3 days)
19. use a 5mL pipette to Triturate cells only 1 or 2 times before seeding.
SECTION: LUHMES Passaging (~ every 2-3 days)
18. Discard the supernatant and resuspend in 1mL LUHMES growth media 
SECTION: LUHMES Passaging (~ every 2-3 days)
17. Transfer cells to 15mL tube and centrifuge for 5 minutes at 1200 RPM
SECTION: LUHMES Passaging (~ every 2-3 days)
16. Neutralize trypsin with 6mL of pre-warmed DMEM/F12
SECTION: LUHMES Passaging (~ every 2-3 days)
15. Add 4mL of the pre-warmed .025% Trypsin/EDTA and place in the incubator for 3 minutes.  


SECTION: LUHMES Passaging (~ every 2-3 days)
13. Remove media and rinse with DPBS
SECTION: LUHMES growth media
12.  
       
 Add recombinant human FGF to media after seeding cells
SECTION: LUHMES coating protocol
6.  
 Size: 100uL, Storage Temperature:  –20 °C in aliquots
1 mg/mL
2 µg/µL
Thaw an aliquot of Poly-L-Ornithine solution at room temperature.
 

SECTION: LUHMES growth media
11. Change (pre-warmed) media every 1-2 days
 

SECTION: LUHMES coating protocol
7. Dilute the Fibronectin in sterile Hank’s Balanced Salt Solution (HBSS). 

Add 2 µL to 998 µL 

SECTION: LUHMES coating protocol
9. Rinse 3 times with HBSS.
SECTION: LUHMES coating protocol
10. Air dry for 15 minutes and add LUHMES growth media. 
SECTION: LUHMES coating protocol
2. Dilute Poly-L-Ornithine solution to 50ug/mL in Nuclease-free water

Add 500 µL of PLO for every 500 µL Nuclease-free water

SECTION: LUHMES coating protocol
3. Add 7mL of the 50ug/mL PLO to a T-75 overnight at RT.
SECTION: LUHMES coating protocol
4. Rinse flask 3 times with bio-grade water.
SECTION: LUHMES coating protocol
5. Allow the flask to air dry for 15 minutes uncapped and standing upright in the hood. (turn on UV)
SECTION: Cryopreservation
20. Label 2mL cryovials with the date, name, FIV#, Qbench number, passage number and cell type.     
 
Add about 1.5 million cells per vial with 1mL freezing media. 

Freezing Media: 
 7mL LUHMES Media
 4uL B-fgf (40ng)
 2mL FBS (20%)
1mL DMSO (1%)
SECTION: LUHMES Passaging (~ every 2-3 days)
14. Add fresh culture media to flask for at least 15 minutes before seeding cells to
allow media to reach normal pH
SECTION: LUHMES coating protocol
8. Place fibronectin coated flask in incubator for 3 hours
SECTION: LUHMES Differentiation
21. Day 0, when the LUHMES are 80% confluent, add differentiation media and incubate overnight.
SECTION: LUHMES Differentiation
22. Day 1, replate cells onto a new poly-l-ornithine and fibronectin-coated plate. Replate cells at a density of 1 million cells per T-75 flask or ~ 400,000 per well of a 6-well.
SECTION: LUHMES Differentiation
23. Replace LUHMES differentiation media every day while differentiation (neurons become mature in    7-days)
TITLE: CODA: shorthand for calling functions | HuBMAP | JHU-TMC
AUTHORS: Kyu Sang Han, Pei-Hsun Wu, Joel Sunshine, Ashley Kiemen, Sashank Reddy, Denis Wirtz

[DESCRIPTION]
To downsample ndpi or svs images to 10x, 5x, and 1x tifs, use this function:          
      create_downsampled_tif_images
or try Openslide in python
 
To calculate registration on the low resolution (1x) images 
1. calculate the tissue area and background pixels using this function:
      calculate_tissue_ws
2. calculate the registration transforms:
     calculate_image_registration
 
To build a 3D tissue volume using sematic segmentation:
1. generate manual annotations in Aperio imagescope
2. apply the deep learning function to train a model and segment the high resolution (5x or 10x) images:
     train_image_segmentation

To apply the registration to segmented images:
                             apply_image_registration

To build a 3D tissue matrix from registered, classified images:
                             build_tissue_volume
 
To build a 3D cell volume containing nuclear coordinates:
1. Build a mosaic image containing regions of many whole-slide images for cell detection optimization:
                             make_cell_detection_mosaic
2. Manually annotate the mosaic image to get the ‘ground-truth’ number of cell nuclei:
                             manual_cell_count
3. Determine cell detection parameters using the manual annotations on the mosaic image:
                             get_nuclear_detection_parameters
4. Deconvolve the high-resolution (5x or 10x) H&E images before applying the cell detection algorithm:
                             deconvolve_histological_images
5. Detect cells on the hematoxylin channel of the high-resolution images:
                             cell_detection
6. Apply the registration to the cell coordinates:
                             register_cell_coordinates
7. Build a 3D cell coordinate matrix corresponding to the 3D tissue matrix:
                             build_cell_volume

[STEPS]
TITLE: Obtain LB broth medium
AUTHORS: An.Huang

[DESCRIPTION]
LB broth medium is a basic culture media for microorganisms. This protocol helps make an ordinary LB broth medium.

[STEPS]
1. Dissolve 25 gLB broth powder in 1000 mL distilled water.



2. Autoclave the medium.
 

3. Store the medium at 4 °C.
TITLE: Atomic Force Microscopy of DNA and DNA-Protein Interactions
AUTHORS: Philip J. Haynes, Kavit H. S. Main, Alice Pyne

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Atomic force microscopy (AFM) is a microscopy technique that uses a sharp probe to trace a sample surface at nanometre resolution. For biological applications, one of its key advantages is its ability to visualize substructure of single molecules and molecular complexes in an aqueous environment. Here, we describe the application of AFM to determine the secondary and tertiary structure of surface-bound DNA, and it’s interactions with proteins.</div><div class = "text-block"><span style = "font-weight:bold;">Methods:</span></div><div class = "text-block"><span>Mica and DNA are both negatively charged at a neutral pH in aqueous solution. There are a number of methods used to facilitate the adsorption of DNA to the mica surface [9, 24, 29-31]. The following sections describe three methods to facilitate DNA and DNA-protein adsorption on mica, which are appropriate for imaging DNA in liquid (</span><span style = "font-style:italic;">see</span><span style = "font-style:italic;font-weight:bold;"> </span><span style = "font-weight:bold;">Note</span><span> 5).</span></div><div class = "text-block"><span>Mica substrates can be attached to a steel disc to be mounted on magnetic sample holders as common in AFM instruments (</span><span style = "font-style:italic;">see</span><span style = "font-style:italic;font-weight:bold;"> </span><span style = "font-weight:bold;">Note</span><span> 6).</span></div></div>

[STEPS]
TITLE:          ELISA for quantification of human C4 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human C4 coating antibody is adsorbed onto the microwells by incubation overnight at 4°C with carbonate-bicarbonate buffer.
?. Add 50 µl of human serum or plasma. Human C4 present in the serum or plasma binds to antibodies adsorbed into the microwells.
?. The microplate is blocked with 3% non-fat milk-PBS buffer and later wash to remove unbound proteins.
?. Fifty (50) µl of biotin-conjugated anti-C4 antibody is added. The optimal dilution must be investigated.
?. The microplate is rewashed with PBS-Tween 20 buffer, pH 7.4.
?. One hundred µl of streptavidin-HRP conjugate is added and it binds to the biotin-conjugated anti-C4 antibody. The optimal dilution of this conjugate must be investigated.
?. The plate is washed following incubation to remove the unbound Streptavidin-HRP.
?. Add 100 µl of 3,3',5,5'- tetramethylbenzidine (TMB; Sigma-Aldrich) into each well.
?. Incubate the microwells in the dark for 15 min.
?. A colored product is formed in proportion to the quantity of C4 present in the sample or standard.
?. The reaction is terminated by addition of 100 µl 3M H2SO4  and the absorbance is measured at 450 nm.
?. A standard curve is made from 7 human C4 standard dilutions and the human C4 sample concentration is determined.