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[danilexn]

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[velmeshevlab]

Thanks so much for a great protocol paper and making everything open-source and so easy to follow! I have a question about sequencing machine model/flow cell: can NextSeq 2000 be used for Open-ST?

[lmschott]

Hi,
Thanks for the kind words! I believe in theory the NextSeq2000 can also be used for Open-ST, but we have never tried it so I have no tips to give. The NextSeq2000 is also patterned and actually has a higher cluster density (~4900 vs 2900 K/mm^2 of the NovaSeq6000). I have tried opening a used NextSeq2000 flow cell before and know that the glue that holds it together is much stronger, so I would suggest practicing that before giving it a try.

[Y-SASAGAWA]

Thank you for publishing detailed information on Open-ST.

I have a question on one of the product you use.
The following products are used in the protocol of the paper.

16-Well ProChamber Microarray System	
Grace Bio-Labs	
Cat#645508

I tried to purchase this product but could not find the catalogue number on the GraceBio-Labs home page (https://gracebio.com/).
If you know the corresponding URL, could you please let me know?

Also, if you know of an alternative product name and its part number, I would be grateful if you could let me know.

Thank you in advance.

[lmschott]

Hi,
It seems that Grace Biolabs has stopped selling this product.

As an alternative we also use:
Grace Bio-Labs ProPlate® microarray system, slide module (GBL204862-2EA), https://www.sigmaaldrich.com/DE/en/product/sigma/gbl204862
or
12 Well Chamber, removable (Cat.No:81201), https://ibidi.com/chamber-slides/46-12-well-chamber-removable.html

Either works with the ~3x4 mm pieces and 100 uL reaction volume, and we seal them with sticky tape.
We reuse the Grace-biolabs product (cleaning with ethanol and RNase away). The ibidi chamber becomes leaky at some point, so I don't like to reuse it.
With these products I have (very rarely) observed some leaking and they are more difficult to seal efficiently. Thus, I like to space out my capture areas and make a humid chamber for my overnight incubation.
Let us know if you have other suggestions :)

[Y-SASAGAWA]

Thank you for your introduction and detailed explanation of alternative products.

I will try these two products first.

It seems that there are various slide chambers available from other companies.

If I find something good, I will give feedback here.

[lmschott]

Thank you!

[Rin-Kawagoe]

Thank you for publishing the detailed information on Open-ST.
I have two questions regarding the protocol:

1. Could you please share the manufacturer and product number of the plate seal used for capture area labeling? I'm concerned that a plastic seal might dissolve in 100% methanol during the fixation step.
2. Are the Hematoxylin and Eosin used in Open-ST certified as RNase/DNase-free?

[lmschott]

Hi!

1. I use SealPlate foil from Excel Scientific (cat# Z369667). You can buy it via Sigma-Aldrich for example. It sticks well throughout, but can also be removed well without leaving any residue.
2. They are not certified as nuclease-free.

[Rin-Kawagoe]

Thank you for your response.
I am concerned that the plate seal might dissolve, so I would appreciate it if you could provide more details about the methanol fixation process.
For example, is the procedure carried out inside the cryostat, or in a fume hood while keeping the sample cooled with dry ice?
Additionally, it would be helpful to know the type of tube (e.g., 1.5 mL, 5 mL, etc.) used to hold the methanol.

[lmschott]

Sure! We fix in methanol for 30 min at -20°C. For each sample I put 1 mL 100% methanol in a 2 mL roundbottom tube and let that pre-cool in a -20°C freezer.
For safety reasons, I would transfer the samples (cryosection on the capture area stuck to sticky tape) on dry ice to a fume hood and then place them in the pre-cooled methanol. Once the sample is in the methanol, I place the closed tubes back in the -20°C.
Hope this helps!

[Rin-Kawagoe]

Thank you for your detailed explanation.
I will try it as you described.