Meet your Parhelia Omnistainer
This page uses some old nomenclature, such as Cover Tile instead of Cover Pad and Support Tile instead of Support Pad
Dear Customer,
Congratulations on the acquisition of Parhelia Omni-Stainer and welcome to the Parhelia community! While getting familiar with an entirely new system for staining can seem daunting, we are here to help you through every step.
This guide will take you through how to use Parhelia Omni-stainer as a standalone instrument as well as in conjunction with protocol automation on Opentrons OT-2.
What’s inside Parhelia Omni-stainer starter kit
Overview and assembly instructions in this video
Omni-stainer C12/S12: your brand-new instrument precision-machined out of aluminum, anodized for durability and chemical resistance
(S12 only): a set of 4 shelves are designed to be inserted into the Omni-stainer S12 and support standard 25x75mm microscopy slides.
Perforated lid for automation (automation bundle) or Solid lid (manual bundle)
Cover Tiles (S12) or Support Tiles (C12) (starter pack, 10 pcs): single-use staining chambers made of temperature-resistant polycarbonate
Perforated filter paper (starter pack, 3 pcs): 3mm thick filter paper that is laminated to a double-sided adhesive and protected with a liner. The paper needs to be adhered to the underside of the perforated lid and pre-wetted before each run. Its purpose is to create a moisture barrier around the sample access holes and thus prevent sample evaporation during long incubations.
Conditioning reagent - 5ml bottle. Parhelia conditioning reagents is a proprietary mix that optimizes the liquid flow. We recommend adding it at 1:100 to every buffer used in the protocol in order to ensure smooth liquid exchange and reduce bubble formation. We noticed however that if it is added at the rehydration step (when a dry tissue sample is first exposed to an aqueous buffer), it may negatively affect the staining results, so we recommend skipping it during the rehydration step, but using at all other steps that involve aqueous buffers.
Thermal sheath. (If ordered, it will be shipped separately). Thermal Sheath is designed to be used in combination with OT-2 Temperature Module and insulate Omni-stainer from the top and the sides while it’s being heated from beneath. A sliding shutter on the Thermal Sheath has a tab that is intended to be operated by the tip of the pipette, opening and closing the sample access wells as needed.
Additional materials needed for the first runs
12-trough reservoir (Corning-Axygen): for large reagents, hosts 12 separate reagents up to 21 mL each
Skirted 96-well PCR plate: for small reagents (antibodies, blockers, dyes, fixatives)
V-shaped polystyrene reservoir for sample loading (Corning)
Pierceable aluminum foil (Corning-Axygen)
Blue dye demo reagents:
PBS buffer
Blue Dye for demo runs and volume calibration. - Bromophenol blue solution, 0.04% in PBS (dissolve 40mg of Bromophenol blue in 100 mL of PBS and shake until dissolved), or ready-made solution from Sigma
Setting up the Opentrons OT-2 (automation bundle only)
Instrument setup
Follow the setup instructions on the Opentrons website. You will need:
A computer that will be connected to the OT-2 instrument
An user account with sufficient permissions to install the Opentrons app
A free Ethernet port (or an Ethernet adapter if you use a laptop)
Import custom labware definitions
Labware definitions are special files in JSON format that contain the information about the geometry of labwares (number of wells, XY positioning, well depth etc) that the Opentrons OT-2 robot relies on to correctly access the samples. We created custom labware definitions that describe Parhelia Omni-stainer instruments and reservoirs (deep-well 12-trough, 96-well black plate and 96-well skirted plate) that we use in our OT-2 protocols. Follow this guide for labware definitions download and import.
Protocol programming
Opentrons protocols are written using the Python programming language and make use of Opentrons’ Python API. This innovative feature of the OT-2 makes good use of Python’s combination of simplicity and expressive power, so you can easily program arbitrarily complex protocols even as a beginner. Follow this comprehensive guide on programming OT-2 with Python.
StainWorks
Since many researchers are not familiar with with Python, we at Parhelia created StainWorks: a simple yet powerful web app that provides two essential features:
Templating - the ability to configure Python protocols without having to dig into the Python code. Instead, the relevant run parameters (number of samples, wash volumes.
Global Library - a curated collection of Python scripts that implement various assays (CODEX, IHC, H&E etc). These protocols have been created, validated and maintained by the Parhelia team.
Labware layouts - Each template in StainWorks comes with a special “Labware Layout” spreadsheet that is attached to the template. Labware Layout contains instructions on how to correctly fill reservoirs with reagents and place them onto the deck of an OT-2 robot for a particular protocol run.
Accessing StainWorks requires having a user account. If you purchased an S12 or C12 automation bundle, your StainWorks account will be set up automatically and a 90-day free trial of StainWorks is included with your purchase. You will receive a separate email from sales@parheliabio.com with your account credentials. You can learn more about exactly how to use StainWorks in the “Blue Dye Demo” tutorial below.
Preparing the Parhelia Omni-stainer for the run
Place Parhelia Omni-stainer on the bench
Pour 10mL of ddH2O to the waste tray at the bottom- this will maintain the humidity inside the instrument and prevent sample evaporation.
Automation bundle only:
Place the lid on the bench, with the underside facing up, make sure it’s clean and dry. Take one perforated blotting paper sheet and remove the liner. Carefully position the adhesive side over the underside of the lid, making sure that the holes in the paper and the lids line up and press it down.
Use the ddH2O squirt bottle to infuse the blotting paper with 3-5 mL of water. Don’t overdo it - the paper needs to be wet, but not soaking.
Load the samples following the instructions below
Sample preparation and loading
Preparing samples
FFPE sections: a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative and re-hydrated. If HIER is needed, at this time we recommend it to be. It is possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath, but that functionality is experimental and hasn’t been fully tested. Please reach out to us directly at info@parheliabio.com if you would like to try running HIER with Parhelia Omni-stainer.
Fresh frozen sections: sample needs to be fixed and re-hydrated
Cell culture/cell spreads: samples needs to be fixed and re-hydrated
Slide-mounted samples
Setup:
Take a Cover Tile and remove the peel tabs, following this video
Mount the cover tile as shown and follow this video
Coverslip-mounted samples:
Setup:
Follow this video for mounting coverslip samples onto Support tiles
Preparing samples
FFPE sections: a sample needs to be baked, deparaffinized with Xylenes or a suitable alternative and re-hydrated. If HIER is needed, at this time we recommend it to be. It is possible to do HIER directly in Parhelia Omni-stainer using the Thermal Sheath, but that functionality is experimental and hasn’t been fully tested. Please reach out to us directly at info@parheliabio.com if you would like to try running HIER with Parhelia Omni-stainer.
Fresh frozen sections: sample needs to be fixed and re-hydrated
Cell culture/cell spreads: samples needs to be fixed and re-hydrated
Ensure that the coverslip is flush with the bottom edge of the support tile and is touching the wicking pillar.
Ensure that there is a capillary coupling between the staining chamber and the pillar (basically, a drop of liquid connecting the two). If that’s not the case, dispense 100ul of PBS mixed with 1ul of Parhelia conditioning reagent onto the contact point between the wicking pillar and the support tile.
Blue Dye Demo
This is a very simple demo protocol that helps you understand how capillary gap exchange works inside Parhelia Omni-stainer and calibrate volumes that are needed for the gap exchange.
Manual run
Prepare two Eppendorf tubes and fill one with PBST and the other one with Blue Dye (1.0 mL each). Add 10ul of Parhelia Conditioning reagent to each, and mix thoroughly.
Using P200 pipette, slowly dispense 110 ul of Blue Dye onto the top curved edge of the S12 covertiles (for C12 coverslips dispense 60ul the solution onto the ramp). Carefully observe how the Blue Dye is drawn into the chamber. Ensure that the clear liquid that was in the staining chamber gets displaced completely and drips down the wicking pillar. Next, wash the staining chamber by dispensing 150ul of the clear buffer. Ensure that the blue dye gets displaced completely. Watch these videos for reference:
Omni-Stainer C12 Manual demo
Omni-Stainer S12 Manual demo
Very small pockets of carryover buffer towards the bottom corners of the staining chamber are normal and should not affect staining quality because usually there’s no tissue present there. However, if you wish for a more complete exchange, you may wish to increase the wash volume as necessary.
If the flow seems abnormally slow, and the exchange seems incomplete and uneven:
check for bubbles in the chamber. If bubbles are present, slide the sample up and down to release the bubble. If that doesn’t help, re-mount the sample.
Make sure that the sample is positioned correctly within the staining chamber. For coverslips, make sure that the coverslip rests evenly on both spacers.
Automated run
Open StainWorks
Download the ‘Blue Dye Demo labware layout.xlsx’ file and follow the instructions inside for filling the labwares, also watch this video.
Place the labwares on the deck according to the Labware Layout, making sure that the recessed corner is in the northwest (top-left) position, also refer to this video.
Take a blank microscopy slide (for S12) or coverslip (for C12), wipe it clean and dry and mount it with a cover tile/support tile according to the directions above. Place it into the position #1 (the one closest to the recessed corner) of the Parhelia Omni-stainer.
In StainWorks, press “modify protocol”
In the dialogue that pops up, select the type of your Omni-stainer (C12 or S12), keep all other values default.
Press “Save and Download”
Upload the .py file that you just downloaded into the Opentrons app.
Close the lid of the Omni-stainer. Visually locate the calibration mark, which looks like + sign embossed on the lid (note that for C12 and S12, the marks are in different locations). Proceed to run the protocol calibration with the lid closed, following the instructions in the video.
If calibration looks good, proceed with the protocol run.
CAUTION: how to correctly re-run the protocol:
Just clicking “Run again”, then “Start Run” will not apply your saved calibration data and the pipetting locations could be off.
To re-run a previously loaded protocol, select “Protocols” on the left side, choose the protocol, then select Run protocol, choose the OT-2 robot and select “Proceed to Setup”.
On the Setup tab click open "STEP 1 Robot Calibration", click Proceed to labware setup. A pop-up window will appear will the calibration data, click Apply stored data, click “Proceed to Run”.
Click “Start run”
Remove the lid and run the protocol to observe the exchange. This is what is should look like:
Parhelia Omni-stainer S12 blue dye demo
Parhelia Omni-stainer C12 blue dye demo
Ensure that the blue dye filling and washing is complete
Very small pockets of carryover buffer towards the bottom corners of the staining chamber are normal and should not affect staining quality because usually there’s no tissue present there. However, if you wish for a more complete exchange, you may wish to increase the wash volume as necessary.
If the flow seems abnormally slow, and the exchange seems incomplete and uneven:
Carefully check for bubbles in the chamber. Bubbles may form if the buffers used have significant amounts of dissolved gas.If bubbles are present, slide the sample up and down to release the bubble. If that doesn’t help, re-mount the sample. When in doubt, degas buffers before the run.
Make sure that Parhelia Buffer Conditioner has been added at 1:100 to both buffers
Slide staining: ensure the gray aluminum shelf is correctly positioned in the staining chamber, if this is off the liquid may be applied to the outside of the cover tile rather than into the capillary gap.
Temperature-controlled protocols
Set up the temperature module on the deck of the Opentrons following this tutorial. We recommend placing the temperature module in position 7 of the OT-2 Deck
Place thermal sheet on top of the Omni-Stainer
When running the protocols, make sure to select “omni_stainer_###_with_thermosheath”, where ### is the instrument model (i.e. S12_slides or C12_cslps)
During the calibration step, position the pipette tip over the calibration pin
If you are writing your own protocol, make sure to use functions openShutter(...), closeShutter(...) that you can find in the protocol header. Failure to do so may get your pipette damaged and we are not going to be responsible.
Besides this email, we have a documentation page that we are actively updating and expanding: https://parhelia-biosciences.gitbook.io/omni-stainer-user-guide/
If you identify any missing bits that would be good to cover, please reach out to me or the team at support@parheliabio.com.
Yours sincerely,
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