Embedding cells in multiwell plates

[Glen Tibbits]

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This is an EM and not a confocal question but the expertise of this group is sought.

 

We want to fix and embed cells for EM in a 48 well polystyrene plate.  The plate cannot be changed to a different material.  We are looking for an epoxy that is compatible with both doing EM and the polystyrene material (i.e. not acetone soluble).  Ideally we would fix and embed the cells in each well and then remove the epoxy discs for sectioning.

 

Any input would be appreciated.

 

Thanks

 

Glen Tibbits, PhD

Professor  | Biomedical Physiology & Kinesiology | Simon Fraser University,

Member    | Centre for Cell Biology, Development, and Disease | Simon Fraser University

Senior Scientist  | BC Children’s Hospital Research Institute

604 910 4358

 

I respectfully acknowledge that SFU is on the unceded ancestral and traditional territories of the səl̓ilw̓ətaʔɬ (Tsleil-Waututh), Sḵwx̱wú7mesh Úxwumixw (Squamish), xʷməθkʷəy̓əm (Musqueam) and kʷikʷəƛ̓əm (Kwikwetlem) Nations.

 

[Janssen, William]

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I don't really think the selection of embedding resin is the issue of concern.  The Propylene Oxide is the most immediate problem, and since this sounds like cell culture of a monolayer, or at a maximum 2-3 cell layer thick material, I would simply skip the PO step and go from 100% ethanol dehydration to your resin.

Be forewarned.... getting embedded blocks out of 48 well chambers is tough. Good luck...

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Subject: Embedding cells in multiwell plates

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*****

This is an EM and not a confocal question but the expertise of this group is sought.



We want to fix and embed cells for EM in a 48 well polystyrene plate.  The plate cannot be changed to a different material.  We are looking for an epoxy that is compatible with both doing EM and the polystyrene material (i.e. not acetone soluble).  Ideally we would fix and embed the cells in each well and then remove the epoxy discs for sectioning.



Any input would be appreciated.



Thanks



Glen Tibbits, PhD

Professor  | Biomedical Physiology & Kinesiology | Simon Fraser University,

Member    | Centre for Cell Biology, Development, and Disease | Simon Fraser University

Senior Scientist  | BC Children’s Hospital Research Institute

604 910 4358



I respectfully acknowledge that SFU is on the unceded ancestral and traditional territories of the səl̓ilw̓ətaʔɬ (Tsleil-Waututh), Sḵwx̱wú7mesh Úxwumixw (Squamish), xʷməθkʷəy̓əm (Musqueam) and kʷikʷəƛ̓əm (Kwikwetlem) Nations.

[Barbara Smith]

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We do embed cell culture in plates routinely. Do not use Propylene Oxide , go straight from 100% ethanol changes (we do 3 5-minute changes) to your resin (we use EPON). For the first overnight change, use resin without catalyst and then three changes the next day of 100% resin with catalyst.  Do NOT go straight to a 60C oven; go to a 37C oven for 2-3 days, check to see if plastic is "tacky" and then  transfer to 60C.  As an added note, for smaller wells, I fill up the well with more EPON and then pop out of the plastic and use a saw to cut the sample into multiple blocks.


Hope this helps,

Barbara


Barbara J Smith

EM Specialist

JHU SOM Microscope Facility

Baltimore, MD 21205


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From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Tuesday, April 21, 2020 12:01 PM
To: [hidden email]
Subject: Embedding cells in multiwell plates


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*****

This is an EM and not a confocal question but the expertise of this group is sought.



We want to fix and embed cells for EM in a 48 well polystyrene plate.  The plate cannot be changed to a different material.  We are looking for an epoxy that is compatible with both doing EM and the polystyrene material (i.e. not acetone soluble).  Ideally we would fix and embed the cells in each well and then remove the epoxy discs for sectioning.



Any input would be appreciated.



Thanks



Glen Tibbits, PhD

Professor  | Biomedical Physiology & Kinesiology | Simon Fraser University,

Member    | Centre for Cell Biology, Development, and Disease | Simon Fraser University

Senior Scientist  | BC Children’s Hospital Research Institute

604 910 4358



I respectfully acknowledge that SFU is on the unceded ancestral and traditional territories of the səl̓ilw̓ətaʔɬ (Tsleil-Waututh), Sḵwx̱wú7mesh Úxwumixw (Squamish), xʷməθkʷəy̓əm (Musqueam) and kʷikʷəƛ̓əm (Kwikwetlem) Nations.

[Carol Heckman]

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We wrote a protocol about this.  If you search Heckman and TEM in Nature Protocols, it should come up.
Carol Heckman
Bowling Green State University
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Sent: Tuesday, April 21, 2020 12:26 PM
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Subject: [EXTERNAL] Re: Embedding cells in multiwell plates

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I don't really think the selection of embedding resin is the issue of concern.  The Propylene Oxide is the most immediate problem, and since this sounds like cell culture of a monolayer, or at a maximum 2-3 cell layer thick material, I would simply skip the PO step and go from 100% ethanol dehydration to your resin.

Be forewarned.... getting embedded blocks out of 48 well chambers is tough. Good luck...

________________________________
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Sent: Tuesday, April 21, 2020 12:01:10 PM
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Subject: Embedding cells in multiwell plates

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*****

This is an EM and not a confocal question but the expertise of this group is sought.



We want to fix and embed cells for EM in a 48 well polystyrene plate.  The plate cannot be changed to a different material.  We are looking for an epoxy that is compatible with both doing EM and the polystyrene material (i.e. not acetone soluble).  Ideally we would fix and embed the cells in each well and then remove the epoxy discs for sectioning.



Any input would be appreciated.



Thanks



Glen Tibbits, PhD

Professor  | Biomedical Physiology & Kinesiology | Simon Fraser University,

Member    | Centre for Cell Biology, Development, and Disease | Simon Fraser University

Senior Scientist  | BC Children’s Hospital Research Institute

604 910 4358



I respectfully acknowledge that SFU is on the unceded ancestral and traditional territories of the səl̓ilw̓ətaʔɬ (Tsleil-Waututh), Sḵwx̱wú7mesh Úxwumixw (Squamish), xʷməθkʷəy̓əm (Musqueam) and kʷikʷəƛ̓əm (Kwikwetlem) Nations.

[Julia Edgar]

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Presumably the cells cannot be grown on glass coverslips that can then be removed from the dish. I understand there is a method to remove the glass after embedding everything in resin.
Best wishes
Julia

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From: Confocal Microscopy List <[hidden email]> on behalf of Carol Heckman <[hidden email]>
Sent: Tuesday, April 21, 2020 6:45:49 PM
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Subject: Re: Embedding cells in multiwell plates

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*****

We wrote a protocol about this.  If you search Heckman and TEM in Nature Protocols, it should come up.
Carol Heckman
Bowling Green State University
________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of Janssen, William <[hidden email]>
Sent: Tuesday, April 21, 2020 12:26 PM
To: [hidden email] <[hidden email]>
Subject: [EXTERNAL] Re: Embedding cells in multiwell plates

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*****

I don't really think the selection of embedding resin is the issue of concern.  The Propylene Oxide is the most immediate problem, and since this sounds like cell culture of a monolayer, or at a maximum 2-3 cell layer thick material, I would simply skip the PO step and go from 100% ethanol dehydration to your resin.

Be forewarned.... getting embedded blocks out of 48 well chambers is tough. Good luck...

________________________________
From: Confocal Microscopy List <[hidden email]> on behalf of [hidden email] <[hidden email]>
Sent: Tuesday, April 21, 2020 12:01:10 PM
To: [hidden email]
Subject: Embedding cells in multiwell plates

USE CAUTION: External Message.

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*****

This is an EM and not a confocal question but the expertise of this group is sought.



We want to fix and embed cells for EM in a 48 well polystyrene plate.  The plate cannot be changed to a different material.  We are looking for an epoxy that is compatible with both doing EM and the polystyrene material (i.e. not acetone soluble).  Ideally we would fix and embed the cells in each well and then remove the epoxy discs for sectioning.



Any input would be appreciated.



Thanks



Glen Tibbits, PhD

Professor  | Biomedical Physiology & Kinesiology | Simon Fraser University,

Member    | Centre for Cell Biology, Development, and Disease | Simon Fraser University

Senior Scientist  | BC Children’s Hospital Research Institute

604 910 4358



I respectfully acknowledge that SFU is on the unceded ancestral and traditional territories of the səl̓ilw̓ətaʔɬ (Tsleil-Waututh), Sḵwx̱wú7mesh Úxwumixw (Squamish), xʷməθkʷəy̓əm (Musqueam) and kʷikʷəƛ̓əm (Kwikwetlem) Nations.