eamag/protocols-with-steps huggingface dataset:
TITLE: TAB-PAINT imaging of alpha-synuclein fibrils using Nile Red
AUTHORS: Ezra Bruggeman

[DESCRIPTION]
This is a protocol for the preparation of a sample of alpha-synuclein fibrils on a PLL-coated cover glass for TAB-PAINT imaging with Nile Red. This protocol was used to generate the data shown in Figure 3 of the following publication:

Bruggeman et al., POLCAM: Instant molecular orientation microscopy for the life sciences. bioRxiv 2023.02.07.527479 (Feb 2023), doi: https://doi.org/10.1101/2023.02.07.527479

[STEPS]
SECTION: Protocol
1. Prepare alpha-synuclein fibrils by diluting alpha-synuclein monomer 70 micromolar (µM) in PBS (with 0.01% NaN3) and incubating at 37 °C in a shaker (200 rpm) for over 1440 min. You can store the fibrils at 4 °C for later use.

SECTION: Protocol
2. Argon plasma clean cover glass (VWR collection, 631-0124) for 30 min in a plasma cleaner (Expanded Plasma Cleaner, PDC-002, Harrick Plasma).
SECTION: Protocol
3. In the meantime:
Filter phosphate-buffered saline (PBS) using a 0.02 μm syringe filter (6809-1102, Whatman).
Dilute Nile Red in filtered PBS to a concentration of 1 nanomolar (nM)
 

SECTION: Protocol
4. Create a PLL-coated sample well on the cover glass:
SECTION: Protocol
5. Add fluorescent beads for lateral drift correction:
SECTION: Protocol
6. Use a pipet to remove the excess PBS from the well, add 50 µL of alpha-synuclein fibrils and pipet up and down a few times in each corner of the sample well and wait for 5 min
 

SECTION: Protocol
4.1. Create a sample well on the cleaned cover glass by sticking a frame-seal slide chamber (9x9 mm, SLF0201, Bio-rad) on the glass.
SECTION: Protocol
4.2. Pipet 70 µL of 0.01% PLL (0.01% poly-L-lysine solution, P4707, Sigma-Aldrich) into the well and wait for 15 min. The PLL will coat the surface of the cover glass.
 

SECTION: Protocol
4.3. Use a pipet to remove the excess PLL from the well and immediately replace it with 70 µL of filtered PBS.
SECTION: Protocol
4.4. Use a pipet to remove the excess filtered PBS from the well and replace with70 µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.
SECTION: Protocol
5.1. Remove the excess PBS from the well using a pipet.
SECTION: Protocol
5.2. Using a pipet, add 50 µL of 0.1 µm diameter TetraSpeck Microspheres (0.1 µm, fluorescent blue/green/orange/dark red, T7279, Invitrogen) into the well.
 

SECTION: Protocol
5.3. Gently pipet up and down a few times and wait for 5 min.

SECTION: Protocol
5.4. Use a pipet to remove the excess solution from the well and replace with70 µL filtered PBS. Gently pipet up and down in the corners of the well. Repeat this step 2 more times.
SECTION: Protocol
7. Remove excess solution using a pipet and add 50 µL of filtered PBS to the well.
SECTION: Protocol
8. Remove excess solution again and replace by 50 µL of 1 nanomolar (nM) Nile Red.
SECTION: Protocol
9. Image the sample straight away and make sure it doesn't dry out during imaging.
TITLE: Immunostaining of Bodo saltans
AUTHORS: Ewa  Chrostek,   Mastaneh Ahrar,   Gregory Dd Hurst

[DESCRIPTION]
This protocol is used in our Laboratory in Liverpool to perform IF on Bodo saltans cells. 

[STEPS]
SECTION: Culture conditions
1. Bodo saltans was cultured in a cerophyl-based medium enriched with 3.5 mM sodium phosphate dibasic (Na2HPO4)1. Cultures were incubated at 22 °C in T25 tissue culture flasks containing 20 ml of media bacterized with Klebsiella pneumoniae subsp. Pneumoniae (ATCC® 700831). 
SECTION: Immunostaining
2. Please, follow the steps below to prepare cells for immunostaining.
SECTION: Immunostaining
2.1. Filter the culture through 100 and 8 µm filter.
SECTION: Immunostaining
2.2. Harvest the cells by centrifugation at 1200 × g for 12 mins at 19 °C.
SECTION: Immunostaining
2.3. Wash the cells with PBS and centrifuge as described above. 
SECTION: Immunostaining
2.4. Dissolve the pellet in 15 µl of PBS and mix with the same volume of low melting temperature agarose (eg. Thermo Fisher Scientific) in a single well of a 96-well plate. Let it ser for a few seconds.
SECTION: Immunostaining
2.5. Add 200 µl of 4% PFA and incubate at room temperature for 10 minutes or at 4 °C overnight.
SECTION: Immunostaining
2.6. Wash with PTX (PBS + 0,1% TritonX) 4 times, 30 mins each wash.
SECTION: Immunostaining
2.7. Block in 5% FBS+PTX overnight at 4 °C.
SECTION: Immunostaining
2.8. Incubate with primary antibody (1:100, diluted in PTX + 5% FBS), for 5 hours at room temperature or overnight at 4 °C. 
SECTION: Immunostaining
2.9. Rinse and wash 3 times for 1 hour with PTX at room temperature. 
SECTION: Immunostaining
2.10. Incubate with secondary antibody (1:1000, diluted in PTX + 5% FBS) overnight at 4°C.
SECTION: Immunostaining
2.11. Rinse and wash 3 times for 1 hour with PTX at room temperature. During 2nd wash add Hoechest 33342 (Thermo Fisher, 1:2000) for 10 minutes. This will be rinsed away with the 3rd wash.
SECTION: Immunostaining
2.12. Remove the agarose from the well with clean forceps and place it on a microscope slide. 
SECTION: Immunostaining
2.13. Add a drop of a mounting medium (eg. Vectashield, Vector Laboratories), and flatten the agarose as much as you can using the coverslip. 
SECTION: Immunostaining
2.14. Proceed with either fluorescence or confocal imaging.
SECTION: Immunostaining
2.15.   

SECTION: Immunostaining
2.16.   

SECTION: References
3. Gomaa Fatma, Li ZuHong, Docampo Roberto, Girguis Peter, E. V. Bodo saltans culture protocol V.2. Protocols.io (2018). doi:10.17504/protocols.io.sh6eb9e
TITLE: Pt_Cas9-2A-ShBle, g24739-A, mRFP, g24739-B
AUTHORS: Mark Moosburner

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">I am creating this protocol to deposit a plasmid sequence and map. </div><div class = "text-block">The episome contains Cas9-2A-ShBle expression cassette and 2 sgRNA expression cassettes targeting the gene Phatr3_J24739. </div></div>

[STEPS]
TITLE: PIP MRI Magnetization Transfer Ratio (MTR) Measurement of Myelofibrosis in Mouse Tibia
AUTHORS: Thomas L Chenevert

[DESCRIPTION]
The goal of this Co-Clinical Imaging Research Program (CIRP) pre-clinical imaging protocol (PIP) is to provide detailed description of key steps used to achieve a stated level of technical repeatability (precision) embodied in “Claims”, for MRI measurement of magnetization transfer ratio (MTR) in tibia bone marrow of myelofibrosis mouse models. This pre-clinical imaging procedure document will be referred to as a “profile” and adheres to a PIP MRI template provided in: https://www.protocols.io/workspaces/pre-clinical-imaging-protocols. 
Here we treat MTR as a “quantitative” metric, although numerical MTR results are a strong function of multiple experimental parameters and MRI hardware conditions. This profile will adopt the common practice to express MTR values in on a 0 to 1 scale. Empirically, MTR reflects fraction of visible signal loss due to application of off-resonance saturation RF energy depleting magnetization in the semi-solid (invisible) macromolecular matrix thus reducing magnetization replenishing visible signal.Simple fluids free of macromolecules should have an MTR » 0, whereas macromolecule-rich semi-solid tissues such as myelin, muscle, scar and collagen exhibit relatively high MTR (> 0.5). This document details procedures for MTR measurement in MF mouse tibia to achieve stated performance claims.
Tibia bone marrow composition in MF mouse models has gradation going from proximal to distil ends of the tibia, therefore separate claims are made for volume of interest (VOI) analysis of MTR maps for each of three distinct sections along the length of the tibia (see Figure 1):
Section 1 (proximal)º VOI (~4-5mm3) within 9mm of proximal end of tibia
Section 2 (transition)º VOI (~0.4-0.5mm3) from 10 to 12mm of proximal end of tibia
Section 3 (distil)º VOI (~0.1-0.2mm3) from 13 to 14mm of proximal end of tibia

Claim 1:A measured change in the mean MTR in Section 1 VOI of MF mouse model tibia that exceeds ±0.16 indicates a true biological change has occurred in the tibia bone marrow with 95% confidence.
Claim 2:A measured change in the mean MTR in Section 2 VOI of MF mouse model tibia that exceeds ±0.11 indicates a true biological change has occurred in the tibia bone marrow with 95% confidence.
Claim 3:A measured change in the mean MTR in Section 3 VOI of MF mouse model tibia that exceeds ±0.09 indicates a true biological change has occurred in the tibia bone marrow with 95% confidence.

[STEPS]
TITLE: Open Field Protocol
AUTHORS: Michael Lee

[DESCRIPTION]
This protocol details about Open Field procedure.

[GUIDELINES]
Detailed records should be kept on every experiment, noting date, timing, subject test order, procedural details followed and any relevant observations pertaining to the assay (e.g. specific behavior or procedural errors).

[STEPS]
SECTION: Set up
1. 20 x 20 x 10 inch chamber (LxWxH).
SECTION: Test
6. Record locomotor activity and track how much time the animal spends in each zone for 30 min.

SECTION: Set up
2. Opaque chambers or dividers between chambers.
SECTION: Set up
4. Have chambers on enclosed rack to diminish outside light.
SECTION: Set up
3. Light intensity = 250 lux.
SECTION: Test
5. AnyMaze
SECTION: Test
5.1. Set two zones in the chamber. Outer ~3.5 inch perimeter of box is one zone and the center of the maze is the other zone.
SECTION: Test
7. Wipe down chambers with ethanol between animals.
SECTION: Test
8. Run males before females if running both genders on same day.
SECTION: Test
9. Can be used as a simple locomotion test by tracking the distance traveled and speed.
SECTION: Test
10. Can also be used as an anxiety test. Less anxious animals will spend more time in the center of the maze.
TITLE: Optical Density Analysis for DAB Staining of Mouse Brain Sections
AUTHORS: Katerina Rademacher, Ken Nakamura

[DESCRIPTION]
This protocol describes steps for optical density analysis of immunohistochemical 3,3′-Diaminobenzidine (DAB) staining of mouse brain sections using ImageJ.

[STEPS]
1. Open ImageJ, then calibrate for analysis.
1.1. See https://imagej.net/ij/docs/examples/calibration/ for calibration setup steps and files.
1.2. Select Analyze > Calibrate, then select the following:
i.	Rodbard (NIH Image)
ii.	Unit: O.D.
iii.	Open the calibration.txt file
iv.	Uncheck ‘show plot’
v.	Check ‘Global calibration’
2. Open an image and outline the regions of interest, including a background area for subtraction.
3. Select ‘multi measure’ and copy the results to an Excel sheet for analysis. Repeat for all images.
4. Take the ‘mean OD’ value for each region and subtract the background area ‘mean OD’ value. Values can be plotted directly or normalized to control groups.
TITLE: Western blotting in Chlamydomonas reinhardtii
AUTHORS: João Vitor Molino

[DESCRIPTION]
This protocols describe the steps to perform a western blot in Chlamydomonas reinhardtii cell lysate and supernatant samples.

[BEFORE_START]
Check antibody dilutions
Check buffers disponibility
Prepare 5% milk solution

[GUIDELINES]
Prepare cell material by cultivating in liquid media. 

[STEPS]
SECTION: Sample preparation - Supernatant
1. Centrifuge algae culture at 2000xg for 10 min
Recover supernatant 
10 min
SECTION: Sample preparation - Lysate
2. Centrifuge algae culture at 2000xg for 10 min
Remove supernatant, and re-suspend cells in lysis buffer (50 mM Tris·HCL (pH 8.0), 0.1% Triton X-100), concentrating cells 100-fold.
10 min
SECTION: Sonication
3. Sonicate using appropriate sonication tip. Duty cycle: 0.5 | Cycle: 1.0 s | Amplitude: 20% | Duration: 30 s
Centrifuge for 15 min at 20000xg to remove cells debries and recover soluble proteins
Quantificate soluble protein
15 min
SECTION: Gel electrophoresis | SDS-PAGE
4. Load 30 µg of total soluble protein (TSP) per lane in a 12% SDS-PAGE 
Transfer proteins to a nitrocelulose membrane
Block the membrane with 5% milk solution
Probe the desired protein with the specific antibodie, diluted in 5% milk solution
Wash 2 times with TBST (0.2 M Tris, 1.37 M NaCl, 0.1% Tween-20, pH 7.6)
Add secondary antibody if required
30 4
TITLE: Flow Cytometer Fluorescence Voltration for FCMPASS
AUTHORS: Joshua A Welsh, Sean M Cook, Jennifer Jones

[DESCRIPTION]
Protocol to perform flow cytometer voltration to identify optimal detector settings for small particle analysis. Data acquired from this protocol are compatible with semi-automated analysis tools built into FCMPASS software.

[STEPS]
SECTION: Sample preparation
1. Vortex   bottle on a high setting for 5 s. 

SECTION: Sample preparation
2. Pipette 500 µL of   to two   . Label one tube 'DPBS" and the second tube 'Beads'.

 

SECTION: Sample preparation
3. Add 3 drops of   to the 'Beads'   and vortex for r 5 s. 

SECTION: Cytometer Setup
4. Ensure cytometer is clean and that -Height and -Area statistics are set to be collected on all parameters and that all parameters are on.
SECTION: Cytometer Setup
5. Create a pseudocolor plot with FSC-A on the X-Axis and (488 nm) B-SSC-A on the Y-Axis and make sure both parameters are being plotted on a linear-scale.
SECTION: Cytometer Setup
6. Create a histogram plot with (405 nm) V-SSC-H on the X-Axis and make sure it is plotted on a log-scale.
SECTION: Cytometer Setup
7. Set the cytometer triggering threshold to (405 nm) V-SSC-H. All samples should be acquired with the lowest flow rate, typically ~10-15 µL min-1.
 

SECTION: Cytometer Setup
8. Acquire the DPBS tube while viewing the histogram plot from  . Adjust the detector gain or trigger threshold until the instrument noise is being acquired at ~1000 events/sec. The instrument noise floor is distinct from detected background events in sheath as it has a sharp increase. In a system with debris there may be a tail that elongates out of this this sharp peak. 
  

SECTION: Cytometer Setup
8.1. Recording this noise is not necessary as this step is identifying optimal settings.
SECTION: Cytometer Setup
9. Acquire the 'Beads' tube from   . Using the plot from    adjust the FSC and B-SSC gain until the single bead population is clearly visible and can be easily gated from the doublet population to the top right of it. Use the Violet SSC trigger settings identified in   
 
  

SECTION: Cytometer Setup
10. Creating a gate around the single bead population named 'Gate 1';
SECTION: Cytometer Setup
11. Adjust the stopping criteria of the instrument to record until 10,000 events are acquired on 'Gate 1' drawn in   .

SECTION: Performing Voltration
12. Voltration can now be performed by recording the 'Beads' tube at multiple fluorescent detector gains, leaving the trigger threshold and light scatter gains consistent. It is recommended that a recording of at least 10 fluorescent detector settings is taken. Including more increments within a voltration will result in being more confident of the subsequent optimal detector settings.  
 

SECTION: Performing Voltration
12.1. To ensure accurate data analysis, the brightest bead must be visible on at least two of the selected gains.
TITLE: Coupling of TMEM192 antibody to MyOne™ Epoxy Dynabeads™
AUTHORS: Pawel Lis, Daniel Saarela, Dario R Alessi

[DESCRIPTION]
This protocol describes coupling of 600 µg of rabbit monoclonal TMEM192 antibody (Abcam recombinant Anti-TMEM192 antibody [EPR14330-67], BSA and Azide free, ab232600) to 20 mg of MyOne™ Epoxy Dynabeads™ (Invitrogen™, 34001D) to obtain 2 mL of final suspension. The coupled beads generated using this protocol can be used for the isolation of untagged lysosomes from cells and tissues.


[GUIDELINES]
™
 


[STEPS]
SECTION: Protocol
1. 
Before opening the vial containing dried magnetic beads, equilibrate to Room temperature.

SECTION: Protocol
2. Thaw the antibody on ice and keep on ice until it is needed in step 8.

SECTION: Protocol
3. Calculate the volume of antibody needed, so that 600 µg is used – this volume should
be ≤500 µL.

SECTION: Protocol
4. Weigh 20 mg beads directly into a fresh low-binding 1.5 ml Eppendorf tube.

SECTION: Protocol
5. Resuspend beads in 1000 µL of sterile Milli-Q water, vortex for 15 s, sonicate in a water bath sonicator for 5 min.

SECTION: Protocol
6. Place vial on a magnetic rack for 1 min, remove water using a pipette.

SECTION: Protocol
7. Repeat steps 5 and 6. After sonication there should be no bead aggregates visible.
SECTION: Protocol
8. Add the required volume of antibody to the vial containing washed beads.
SECTION: Protocol
9. Add buffer C1 up to total volume of 500 µL (C1= 500 - antibody volume). Vortex to resuspend the beads.

SECTION: Protocol
10. Add 500 µL of buffer C2 and vortex.

SECTION: Protocol
11. Incubate in a Thermomixer at 37 °C for 16–24 hours (typically 1200 min) at   (make sure the beads do not settle).

SECTION: Protocol
12. Place on a magnetic rack for 1 min, remove liquid using a pipette.

SECTION: Protocol
13. Resuspend beads in 1000 µL of buffer HB, vortex.

SECTION: Protocol
14. Place on a magnetic rack for 1 min, remove liquid using a pipette.

SECTION: Protocol
15. Resuspend beads in 1000 µL of buffer LB, vortex.

SECTION: Protocol
16. Place on a magnetic rack for 1 min, remove liquid using a pipette.

SECTION: Protocol
17. Resuspend beads in 1000 µL of buffer SB, vortex.

SECTION: Protocol
18. Repeat steps 16 and 17:
Place on a magnetic rack for 1 min, remove liquid using a pipette.
Resuspend beads in 1000 µL of buffer SB, vortex.
SECTION: Protocol
20. Resuspend beads in 1000 µL of buffer SB, vortex.

SECTION: Protocol
21. Incubate in a shaker at Room temperature for  

SECTION: Protocol
22. Place on a magnetic rack for 1 min, remove liquid using a pipette.

23. Resuspend beads in 1000 µL of buffer SBS. At this stage beads are at 20 mg/ml and should be stored in the fridge. Beads can be further diluted with buffer SBS to 10 mg/ml, which is the usual working concentration.

SECTION: Protocol
19. Place on a magnetic rack for 1 min, remove liquid using a pipette.
TITLE: Hornwort DNA extraction
AUTHORS: Eftychis Frangedakis

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">The gametophytic tissue of hornworts is rich in polysaccharides (Renzaglia et al., 2009) and it also seems to be rich in polyphenolics. Both compounds pose a problem for DNA. </div><div class = "text-block">A modified CTAB protocol was found to be optimal for hornwort genomic DNA extraction (adapted from Porebski et al. 1997). This protocol uses polyvinyl pyrrolidone to remove polyphenolics and contains an extra ethanol precipitation step with a relatively high NaCl concentration compared standard DNA extraction protocols. At NaCl concentrations higher than 0.5 M,polysaccharides remain in solution and they do not co-precipitate with DNA. </div></div>

[STEPS]
?. Grind 0.5-2 g of tissue using mortar and pestle in the presence of liquid nitrogen until finely ground. Transfer frozen ground tissue to a 30 ml tube.
?. Add 10 ml of 60 oC extraction buffer and 100 mg PVP-40/g tissue (5μl of RNAse A (100mg/ml)). Mix by inversion and incubate in a water bath in 60 oC for 30 min.
?. Add 12 ml of chloroform:IAA (24:1) and mix by inversion to form an emulsion.
?. After mixing thoroughly, spin at 10,000 rpm for 10 min at RT.
?. Transfer aqueous phase to a new 30 ml centrifuge tube.
?. Repeat chloroform:IAA extraction to remove cloudiness (PVP) in aqueous phase. Spin at 7,000 rpm for 10 min at RT.
?. Add ½ volume of 5M NaCl to the final aqueous solution recovered. Mix well. Add two volumes of cold (-20 oC) 95% ethanol. Mix by inversion. Place in freezer (-20 oC) for 10 min to accentuate precipitation. Spin at 13,000 rpm for 15 min.
?. (optional) Resuspend in 2 ml of TE and repeat step 7.
?. Pour off supernatant and wash pellet with cold (0 to 4 oC) 70% v/v ethanol. Spin at 13,000 rpm for 5 min, pour off supernatant and dry pellet for 5 min.
?. Dissolve in 50 μl TE (preferably o/n at 4 oC). Transfer to a 1.5 ml tube.
TITLE: Expression and purification of mCherry-OPTN
AUTHORS: Elias Adriaenssens

[DESCRIPTION]
This protocol describes the purification of mCherry-OPTN.

[STEPS]
SECTION: mCherry-OPTN
1. For mCherry-OPTN, clone human OPTN cDNA in a pETDuet-1 vector with an N-terminal  6x His tag follow it by a TEV cleavage site (Addgene #190191).
SECTION: mCherry-OPTN
2. After the transformation of the  pETDuet-1 vector encoding 6xHis-TEV-mCherry-OPTN in E. coli Rosetta pLySS cells, grow cells in 2x TY medium at 37 °C until an OD600 of 0.4 and then continue at 18 °C.
SECTION: mCherry-OPTN
3. Once the cells reached an OD600 of 0.8, induce protein expression with 50 micromolar (µM) IPTG for 960 min at 18 °C. 

SECTION: mCherry-OPTN
4. Collect cells by centrifugation and resuspend in lysis buffer. 
SECTION: mCherry-OPTN
5. Sonicate the cell lysates.

SECTION: mCherry-OPTN
6. Clear Lysates by centrifugation at 18000 rpm, 45 min, 4 °C in a SORVAL RC6+ centrifuge with an F21S-8x50Y rotor (Thermo Scientific).
SECTION: mCherry-OPTN
7. Filter the supernatant through an   filter and load onto a preequilibrated 5 mL His-Trap HP column (Cytiva).
SECTION: mCherry-OPTN
8. After His tagged proteins were bound to the column, wash the column with three column volumes of wash buffer A. 
SECTION: mCherry-OPTN
9. Elute proteins with a stepwise imidazole gradient (30, 75, 100, 150, 225, 300 mM) by increasing addition of buffer B.
SECTION: mCherry-OPTN
10. Pool the fractions at 75-100 mM imidazole that contains the 6xHis-TEV-mCherry-OPTN.
SECTION: mCherry-OPTN
11. Incubate the pooled samples 30 s with TEV protease at 4 °C.
SECTION: mCherry-OPTN
12. After the 6xHis tag was cleaved off, concentrate the protein using a 50 kDa cut-off Amicon filter (Merck Millipore) and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva).
SECTION: mCherry-OPTN
13. Elute the proteins with SEC buffer. 
SECTION: mCherry-OPTN
14. Analyze fractions by SDS-PAGE and Coomassie staining.
SECTION: mCherry-OPTN
15. Pool fractions containing purified mCherry-OPTN.
SECTION: mCherry-OPTN
16. After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen.
SECTION: mCherry-OPTN
17. Store the proteins at -80 °C.
SECTION: mCherry-OPTN
5.1. Sonicate the cell lysates for 30 s. (1/2)
SECTION: mCherry-OPTN
5.2. Sonicate the cell lysates for 30 s. (2/2)
TITLE: DESI imaging mass spectrometry on liver tissue
AUTHORS: Presha  Rajbhandari, Brent    Stockwell, bs  

[DESCRIPTION]
Acquire DESI imaging mass spectrometry data on liver tissue sections at 40µm spatial resolution - to assess distribution of lipids and metabolites within the tissue.

[STEPS]
1. Perform mass calibration of the instrument using Sodium formate and ion mobility using CCS Major Mix using electrospray ionization source.
 

2. Prepare spray solvent: 98:2 methanol:water with 0.1% formic acid and 20pg/µl leucine-enkephalin as lockmass
3. Fill the syringe and start the flow at 1.5µl/min
4. Set the DESI sprayer angle of 75°, nebulizing gas (N2) pressure of 0.3 PSLM
5. Dry the sample slide with tissue section in a vacuum desiccator for 10 minutes
6. Set the sprayer distance from the slide and the inlet tube and optimize signal intensity on a spare tissue section
7. Mark the corners of the slide with a colored marker pen and scan the slide
8. Place the slides on the DESI slide holder
9. Open HDI Imaging software (Waters Corp.) and import the slide image
10. Select the instrument type, slide holder in use, and mark the slide corners to define image coordinates
11. Define the m/z range, polarity and analyzer mode
12. Define the region of interest to image, pixel size and scan rate and export the experiment file
13. Load the samples on Masslynx software and start acquisition using appropriate data acquisition parameters on MS tune page