eamag/protocols-with-steps huggingface dataset:
TITLE: Immunohistochemistry (using floating section)
AUTHORS: Tae-Un Han, Ellen Sidransky

[DESCRIPTION]
- This is an immunohistochemistry protocol used for evaluation of pathology in mouse brain.


- We successfully detected Gfap, Iba1, CD68, TH and alpha-Syn and phospho S129 alpha synuclein protein expression using this
protocol.


- In this protocol, floating sections are mounted on slides first before starting blocking step. In this way, large number of sections can
be stained more efficiently. 




[STEPS]
1.        Mount floating sections onto slides and dry for 30 min (<45 min), While section drying, PAP pen drawing line and dry 5 min.

2.        PBS rehydrate 10 min, Wash with PBS for 5 min three times.

3.        Blocking sol 500 µL, incubate 60 min  at Room temperature.
4.        Replace buffer to primary antibody sol, incubate 10 min at 4 °C.

5.        Wash in PBST three times for 5 min, 10 min and 5 min.

6.        Secondary antibody sol, incubate for 90 min  at Room temperature.

7.        Wash in PBS three times for  5 min, 10 min and 5 min.
8.        Mounting with Vectorshield mounting media
TITLE: Neuropathy Phentoyping Protocols - Nerve Conduction Velocity
AUTHORS: Eva Feldman

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span style = "font-weight:bold;">Summary:</span></div><div class = "text-block"><span style = "font-weight:bold;">Phenotyping of Rodents for the Presence of Diabetic Neuropathy </span></div><div class = "text-block">In man, the development of diabetic neuropathy is dependent on both the degree of glycemic control and the duration of diabetes. Diabetic neuropathy is a progressive disorder, with signs and symptoms that parallel the loss of nerve fibers over time. Consequently, assessments of neuropathy in mice are not performed at one time point, but are characterized at multiple time points during a 6 month period of diabetes. The degree of diabetes is evaluated in 2 ways: tail blood glucose measured following a 6 hour fast and glycated hemoglobin levels. The initial degree of neuropathy is screened using the methods discussed below. Detailed measures of neuropathy are employed when the initial screening instruments indicate a profound or unique phenotypic difference. This document contains protocols used by the DiaComp staff to examine and measure diabetic neuropathy at the whole animal, tissue and cellular levels.</div><div class = "text-block"><span style = "font-weight:bold;">Diabetic Complication:</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://s3.amazonaws.com/pr-journal/bit24kcn.jpg" /></div></div>

[STEPS]
?. [Settings:]
Motor tests: • Duration .02 ms. • Range 25 Ma. • Low frequency filter 1 Hz. • High frequency filter 10 kHz. • Sensitivity 1 mV. • Time 2 ms/div.Sensory test: • Duration .02 ms. • Range 25 mA. • Low frequency filter 1 Hz. • High frequency filter 10 kHz. • Sensitivity 50 µV. • Time 2 ms/div.
?. [Procedure:]
• To confirm the presence of diabetic neuropathy, nerve conduction velocity (NCV) studies will be performed. The animals will be anesthetized with 30/2.5 mg/kg ketamine/xylazine to prevent discomfort. Body temperature will be monitored with a dermal temperature probe and maintained at 32ºC with a warming lamp during NCV. Body temperature will be maintained at 37ºC after NCV using a warming pad to ease animal stress from anesthetic. The nerve studies will last less than 30 min per rat or mouse. The electrodes will be cleaned with 70% alcohol between animals to maintain pathogen-free status. • Sciatic-tibial motor NCV will be determined by stimulating distally at the sciatic notch and distally at the knee via bipolar electrodes with supramaximal stimulation. The Sciatic-tibial motor NCV is calculated using two points of stimulation along the nerve and measuring the resultant latency. Latency is measured from initial onset to maximum negative peak. • Tail motor latency will be determined by stimulating distally along the tail at a recorded distance of 3 cm. Latency is measured from initial onset to maximum negative peak. • Tail sensory NCV will be determined by stimulating proximally along the tail at a recorded distance of 3 cm. Latency is measured from initial onset to maximum negative peak.
?. [Procedure:]
TITLE: Carver et al, Aged Brain Spatial Profiling  - Tissue Processing
AUTHORS: Chase Carver

[DESCRIPTION]
This protocol provides the steps for mouse brain tissue processing and preparation for use in immunohistochemistry and spatial -omic techniques used in Carver et al., "Senescent- and disease-associated microglia are modifiable features of aged brain white matter"

The steps here should be considered as immediately proceeding the protocol for each separate process to correctly stain and image brain sections in the respective techniques.

[STEPS]
SECTION: Perfusion
1. Euthanize mouse with intraperitoneal injection of 32.5mg/ml pentobarbital
SECTION: Perfusion
3. Perfuse transcardially with ice-cold PBS
5. Bisect the two hemispheres of the brain
SECTION: Brain Processing
4. Decapitate mouse and extract brain from skull
SECTION: Perfusion
2. Extract blood via inferior vena cava with a syringe
6. Submerge right hemisphere in 4% PFA in PBS for 24 hours
7. Microdisect left hemisphere hippocampus from hippocampus adjacent white matter, place each tissue in 1 mL TRIzol in ceramic bead tube for downstream RNA processing
SECTION: Brain sections for immunohistochemistry
8. Steps for cutting brain slices for traditional immunofluorescence
SECTION: Brain sections for spatial -omics
9. Steps for cutting brain slices for spatial -omics profiling for GeoMx and CosMx
SECTION: Brain sections for spatial -omics
9.1. Transfer PFA-fixed brain to cold 70% ethanol
SECTION: Brain sections for spatial -omics
9.2. Embed brain in paraffin wax block within 24 hours
SECTION: Brain sections for immunohistochemistry
8.1. In sucrose solutions, brain should initially float and then eventually sink over the time period in each step.
Transfer brain from PFA to 10% sucrose in PBS for 24 hours
Transfer brain from 10% sucrose to 20% sucrose in PBS for 24 hours
Transfer brain from 20% sucrose to 30% sucrose in PBS for 24 hours
SECTION: Brain sections for immunohistochemistry
8.2. Cryopreserve brain hemisphere in 30% glycerol, 30% ethylene glycol, 40% PBS w/v at -80C
SECTION: Brain sections for spatial -omics
9.3. Cut sagittal sections at 5 μm thickness with a sliding microtome

SECTION: Brain sections for spatial -omics
9.4. Place cut paraffin section in deionized water bath heated to 40-42C
SECTION: Brain sections for spatial -omics
9.5. Mount the section on a Superfrost Plus glass slide by placing the slide submerged under the section and pulling upward such that the section is within the correct window for GeoMx or CosMx (see attachment)
SECTION: Brain sections for immunohistochemistry
8.3. Embed brain in Tissue-tek O.C.T. compound and freeze the brain block in a cassette on top of dry ice
SECTION: Brain sections for immunohistochemistry
8.4. Cut sagittal sections at 30 μm thickness on a Leica CM3050 S cryostat at -20C

SECTION: Brain sections for immunohistochemistry
8.5. Place sections in a well plate containing ice-cold PBS + 0.01% sodium azide and maintain plate in 4C environment prior to further processing
SECTION: Brain sections for spatial -omics
9.6. Allow water to drain from slide and dry and bake slide for 30 min at 60C
TITLE:          ELISA for quantification of human C6 in serum or plasma.
AUTHORS: Angel  Justiz-Vaillant, Belkis Ferrer-Cosme

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

[STEPS]
?. An anti-human C6 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 C6 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-C6 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-C6 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 C6 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 C6 standard dilutions and the human C6 sample concentration is determined.
TITLE: PCR (Toehold) Fragment Protocol for a 25ul reaction
AUTHORS: Janet Standeven

[STEPS]
?. Pipette all reaction agents into pcr tube
?. put pcr tube in thermal cycler and set the annealing temperature to 55 degrees celsius
?. Annealing temperatures vary depending on DNA
?. Set elongation time to 30 seconds
?. 1 min/2kb for Q5 High Fidelity → may vary
TITLE: Immunoprecipitation Protocol
AUTHORS: Sam Li

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Immunoprecipitation is a procedure by which proteins or peptides that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics. Immunoprecipitation can also be used to “enrich” a protein population prior to Western Blotting. For example, one can perform immunoprecipitation with a pan-specific antibody against a protein of interest followed by Western blotting with a modification-specific antibody (such as a phospho-specific antibody or an acetylation-specific antibody).</div></div>

[STEPS]
?. [Preparation of antibody-protein A, G, A/G agarose beads:]
Wash protein A, G, A/G agarose beads with cell lysis buffer by pulsing in a microcentrifuge tube (two minutes at 5,000 rpm). Aspirate and discard supernatant. Wash the beads three times with cell lysis buffer.
?. [Preparation of antibody-protein A, G, A/G agarose beads:]
Adjust antibody concentration to 5-10µg/ml in PBS and transfer 500µl of diluted antibody to 5-10µl of agarose beads for each sample.
?. [Preparation of antibody-protein A, G, A/G agarose beads:]
Place the antibody-protein A, G, A/G agarose mix on a shaker and rotate at 4°C for one hour.
?. [Preparation of antibody-protein A, G, A/G agarose beads:]
Spin down the protein A, G, A/G beads for two minutes at 5,000 rpm and wash the antibody-beads three times with cell lysis buffer.
?. [Preparation of cell extract:]
Collect cells and centrifuge at 1200 rpm for 5 minutes at 4°C.
?. [Preparation of cell extract:]
Discard the supernatant and immediately add 800µl of ice-cold lysis buffer to the cells and vortex, then incubate for 30 minutes on ice.
?. [Preparation of cell extract:]
Freeze and thaw the samples with dry ice for two more cycles or sonicate for 15 seconds to ensure the full release of the proteins from the cells.
?. [Preparation of cell extract:]
Spin lysates at 14,000 rpm in a pre-cooled centrifuge for 10 minutes and keep the supernatant.
?. [Immunoprecipitation:]
Adjust the protein concentration of the supernatant to 1-2mg/ml with lysis buffer.
?. [Immunoprecipitation:]
Mix 100-500µl of cell extract with antibody-protein A, G, A/G agarose and rotate the samples at 4°C for about two hours.
?. [Immunoprecipitation:]
Collect the agarose beads by pulsing in a microcentrifuge tube (two minutes at 5,000 rpm, 4°C). Aspirate and discard the supernatant. Wash the beads 3 times with ice-cold cell lysis buffer.
?. [Immunoprecipitation:]
After the final wash, remove the supernatant and add 20µl of 2X SDS sample buffer. Boil for 5 minutes at 95°C and spin down the beads at maximum speed in a microcentrifuge for 5 minutes at room temperature. Carefully pipette off the supernatant.
?. [Immunoprecipitation:]
Load 30µl of sample in each well of a 1.5mm thick gel. Run the gel according to manufacturer’s recommendations and continue with immunoblotting using BioLegend’s Western Blotting protocol (alternately, radiolabeled proteins prepared from target cells can be used to directly visualize the immunoprecipitated protein).
TITLE: Neuronal co-culture
AUTHORS: Nisha Mohd Rafiq, Pietro De Camilli

[DESCRIPTION]
This protocol describes the co-culturing of iPSC-derived dopaminergic (DA) neurons and iPSC-derived medium spiny neurons (MSNs) in a microfluidic compartmentalization device.

[STEPS]
SECTION: Neuronal co-culture device set-up
1. 
 
Coat chambers with 200 µL per well with 0.1 1524 Poly-L-Ornithine (PLO) in PBS.
SECTION: Neuronal co-culture device set-up
2. Incubate plates overnight at 37 °C. 
SECTION: Neuronal co-culture device set-up
3. Wash the chambers thrice with PBS. 
SECTION: Neuronal co-culture device set-up
4. Coat chambers with 200 µL per well 10 1527 Laminin plus 2 1527 fibronectin, both diluted in PBS. 

SECTION: Neuronal co-culture device set-up
5. Incubate plates overnight at 37 °C . Do not store coated plates. Proceed with preparation of plates for seeding cells.

SECTION: Preparation of device for seeding cells
7. Discard coating reagents and add 200 µL per well of NB/B27 complete medium.
Keep the plate at 37 °C for 15 min before seeding cells.
SECTION: Preparation of device for seeding cells
8. Replate cultured iPSC-derived dopaminergic neurons (day 30, see Method section of paper) on one side of the two-chamber microfluidic compartmentalization device (OMEGA4, eNuvio) at a cell concentration of 3×105. Only the axons of DA neurons can migrate through the microfluidic channels connected to the adjacent chamber. 
SECTION: Preparation of device for seeding cells
9. Feed neurons with fresh NB/B27 media every 3 days. Add 10 1527 Laminin to NB/B27 media every 10 days before feeding the neurons.

SECTION: Preparation of device for seeding cells
10. After an additional 25 days in the co-culture device, thaw frozen iPSC-derived medium spiny neurons (MSN) from BrainXell and plate on the other half of the device (where only the axons of DA neurons are present) at a cell concentration of 3x105 cells.
SECTION: Preparation of device for seeding cells
11. Fix the DA-MSN co-cultures till 7-10 days later for immunofluorescence (see Method section of paper). 
SECTION: Preparation of device for seeding cells
6. Prepare enough amount of NB/B27 medium.

For 500 mL

NB/B27 medium (see Method section of paper), add: 

 
 
Warm NB/B27 medium at 37 °C.
Make NB/B27 complete medium by adding:
TITLE: Image Registration of MALDI IMS to Microscopy
AUTHORS: Nathan Heath Patterson, Jamie Allen, Angela  Kruse, allison.b.esselman, ellie.l.pingry, Danielle Gutierrez, Jeff Spraggins

[DESCRIPTION]
Scope:
To process to register MALDI IMS images to different types of microscopy images using IMS MicroLink and WSIREG.

[STEPS]
1. Install Napari with IMS MicroLink plugin using https://github.com/NHPatterson/napari-imsmicrolink
3. Install the WSIREG plugin with Napari https://github.com/NHPatterson/wsireg
5. When complete, all images will be sampled in the same coordinates as the IMS pixel map.
2. Use MicroLink to align laser ablation marks from the post autofluorescence image to the IMS pixel map.
4. Use WsiReg to align other microscopy modalities. These modalities are aligned to the post autofluorescence image.
TITLE: Western Blot
AUTHORS: Haley   Geertsma

[DESCRIPTION]
This protocol is used to western blot proteins-of-interest.

[STEPS]
1. Add 4X Laemmli buffer to protein samples and incubate at 95oC for 7 minutes.
2. Load samples into 12% polyacrylamide gel and run in running buffer at 80-100V for 60-120 minutes.
3. Transfer gel to 0.2μm nitrocellulose membrane at 350mA for 60 minutes at 4oC.
4. Wash membrane with 1X TBS-T then block in 10% milk for 30 minutes at room temperature.
1X TBS-T: 10mM Tris-HCl + 150mM NaCl + 0.5% Tween-20 in H2O, pH 8.0
5. Wash membrane with 1X TBS-T for 4x 5 minutes.
6. Incubate membrane in primary antibody solution overnight at 4oC.
Antibodies diluted to the appropriate concentration in 2% bovine serum albumin in 1X TBS-T with 0.02% sodium azide
7. Wash membrane with 1X TBS-T for 4x 5 minutes.
8. Incubate membrane in secondary antibody solution for 1 hour at room temperature.
Secondary antibody (horseradish peroxidase conjugated) is diluted in 10% milk.
9. Wash membrane with 1X TBS-T for 4x 5 minutes.
10. Rinse ECL Clarity Solution over membrane 20x then image.
11. Once imaging is complete, wash membrane with 1X TBS-T x2 then H2O x2 then let dry at room temperature overnight before storing.
TITLE: How to expose existing R code as a web service
AUTHORS: Sonia García-Ruiz

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol shows how to expose any existing R method as a web service. The code shown here has been used as part of the development of the CoExp Web Application </span><a href="https://rytenlab.com/coexp" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://rytenlab.com/coexp/</span></a><span>. For further information on CoExp Web, please see our paper: </span><a href="https://www.frontiersin.org/articles/10.3389/fgene.2021.630187/full" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.frontiersin.org/articles/10.3389/fgene.2021.630187/full</span></a></div><div class = "text-block"><span>The R methods used in this protocol correspond to the family of CoExpNets R packages (</span><a href="https://github.com/juanbot/CoExpNets" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://github.com/juanbot/CoExpNets</span></a><span>).</span></div></div>

[STEPS]
?. First, we install the R package Plumber. To install the latest stable version from CRAN, we can type the following R command:install.packages("plumber")
?. The next step will consist of creating the API specification to expose our R methods of interest. In this case, we are going to expose the method 'CoExpNets::getNetworkCategories()' from the CoExpNets family of R packages (https://github.com/juanbot/CoExpNets). This step might sound daunting, but it is very straightforward: we only need to generate an R file to include on it all methods we would like to expose through the web API. In this example, the contents of the new API R file (let's call it 'api.R') should look as follows:#' @get /getNetworkCategories #this is the name we will type as part of the URL on our browser when calling the API methodfunction(){ print("getNetworkCategories() GET method has requested!") # a friendly messageCoExpNets::getNetworkCategories() # calling the R method}
?. Once changes have been saved, the next step would consist of running the web API. To do so, we first need to choose an empty port, open an R console and type the following commands:## Code to load CoExpNets librarieslibrary(CoExpNets)library(CoExpROSMAP)CoExpROSMAP::initDb(mandatory=T)## Code to expose the APIlibrary(plumber) pr("/pathtoyourfile/api.R") %>% pr_run(port=8080)
?. Finally, we test the API:curl "localhost:8080/getNetworkCategories"http://localhost:8080/getNetworkCategoriesIn both cases, the response should be similar to:["CoExpROSMAP"]
TITLE: Washing a MinION flowcell
AUTHORS: Kirstyn Brunker

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the steps required to wash a MInION flowcell using Oxford Nanopore's Flowcell Wash kit (EXP-WSH003). The kit contains a DNase I to digest the previous library and unblock pores to prepare the flowcell membrane for another run. </div></div>

[STEPS]
?. Place the tube of Wash Solution A on ice. Do not vortex the tube.
Solution A contains DNase I, which is especially sensitive to physical denaturation. Mixing should only be carried out by gently inverting the tube.
?. Thaw one tube of Wash Solution B at room temperature. Mix thoroughly by vortexing, spin down briefly and place on ice.
?. In a clean 1.5 ml Eppendorf DNA LoBind tube, prepare the following Wash Mix:Component                        VolumeWash Solution A                 Wash Solution B                Mix well by pipetting, and place on ice. Do not vortex the tube.
20 µl
380 µl
?. If you are still sequencing on the flowcell: stop or pause the sequencing experiment in MinKNOW, and leave the flow cell in the device.
?. Ensure that the priming port cover and SpotON sample port cover are closed and remove all fluid from the waste channel using a P1000 pipette.
?. Rotate the flow cell priming port cover clockwise 90° so that the priming port is visible.
?. Check for air between the priming port and the sensor array. If necessary, using a P1000 draw back a small volume to remove any air:Set a P1000 pipette to 200 µlInsert the tip into the priming port, holding the pipette perpendicular to the flowcell to ensure no air gets in the portTurn the wheel anti-clockwise until the dial shows 220-230 µl, or until you can see a small volume of buffer/liquid entering the pipette tip (do not remove too much liquid or you risk exposing the flowcell to air- a few µl is sufficient)Visually check that there is continuous buffer from the priming port across the sensor array
?. Load 400 µl of the prepared Wash Mix into the flow cell via the priming port, avoiding the introduction of air. Do this as slowly as possibly- you may want to use the same method of pipette control as above i.e. turning the dial clockwise to dispense the liquid into the flowcell.
?. Close the priming port and wait for 30 minutes.
You can monitor the digestion by starting or resuming a run in MinKNOW. The number of pores in strand should eventually drop to zero and the number of single pores should increase as they unblock.
TITLE: Glycerol Stock
AUTHORS: Alba Balletbó, Sebastiaan Kuiper

[STEPS]
?. Take  of overnight culture and add it to a sterile Cryotube.
700 µl
?. Add  of sterile  solution and invert 2-3 times.
300 µl
[Glycerol]
?. Place the glycerol stock in -80ºC until needed again.
?. Grow desired culture overnight in    to  media.    Note: Other desired media can also be used, such as M9 minimal media, Terrific broth (TB), etc.
5 ml
[Luria-Bertani (LB)]
TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Salt solution used in preparation of minimal medium for <em>Chlamydomonas reinhardtii.</em></p>
<p> </p>
<p>Source:</p>
<p>Sueoka, N. (1960) <em>Proc. Natl. Acad. Sci. USA</em> <strong>46</strong>, 83-91.</p>
<p>http://www.chlamycollection.org/Sueoka.html</p>

[STEPS]
?. 
?. 
?. 
?.