IUBio Biosequences .. Software .. Molbio soft .. Network News .. FTP

[Chlamydomonas] Re: Chlamy Digest, Vol 75, Issue 2

Heloisa Ciol via chlamy%40net.bio.net (by helociol from gmail.com)
Fri Dec 13 13:18:32 EST 2013


Hello, Sara,

I am so sorry, I completely forgot to answer when you asked me for the
protocol. Sorry I took too long. I translated the protocol to english by
myself, so if you find any english mistakes or if something does not make
sense, please, ask me so I can fix it and explain it for you.

We adapted a protocol for students experiments as well, but some tissues
resulted in better images than others. For Chalmydomonas, some cells seemed
to be a little wrinkled when we did not make a 20% ethanol dehydration
step, and we think the wrinkled cells might be related to it, but we added
this step for protozoan cells and had better images, so we recommend to
extend the dehydration steps, starting with 20% ethanol.

1. Collect the cell and centrifuge at 1000g  for 5 min
2. Ressuspend the pellet in 1 mL phosphate solution (pH 7.4) containing
2.5% glutaraldehyde and incubate at 4°C for 1 hour
3. Discard the supernatant and add 1 mL of phosphate buffer pH 7,4
containing 2,5% of glutaraldehyde. Incubate for 1 hour at 4°C for fixation.
4. Centrifuge the cells at 1000 g for 5 minutes and wash with 0.1M
phosphate buffer (pH 7,4) for 10 minutes. Repeat it three times.
5. Alcoholic dehydration steps:

Ethanol 20%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 30%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 40%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 50%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 60%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 70%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 80%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 90%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 95%          10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 100%         10 min
Centrifuge at 1000 g for 5 minutes
Ethanol 100%         10 min
Centrifuge at 1000 g for 5 minutes

6. Discard the supernatant and add 0,1 mL of HMDS
7. Apply 10 µL of the sample on the microscope cell
8. Cover the sample with gold.

Hope it helps you somehow

Best regards,


2013/12/13 <chlamy-request from oat.bio.indiana.edu>

> Send Chlamy mailing list submissions to
>         chlamy from net.bio.net
>
> To subscribe or unsubscribe via the World Wide Web, visit
>         http://www.bio.net/biomail/listinfo/chlamy
> or, via email, send a message with subject or body 'help' to
>         chlamy-request from net.bio.net
>
> You can reach the person managing the list at
>         chlamy-owner from net.bio.net
>
> When replying, please edit your Subject line so it is more specific
> than "Re: Contents of Chlamy digest..."
>
>
> Today's Topics:
>
>    1. Scanning Electron Microscope (Sara Cline)
>
>
> ----------------------------------------------------------------------
>
> Message: 1
> Date: Thu, 12 Dec 2013 14:28:25 -0600
> From: Sara Cline <sgcline from gmail.com>
> Subject: [Chlamydomonas] Scanning Electron Microscope
> To: chlamy from magpie.bio.indiana.edu
> Message-ID:
>         <
> CAKXojFVa9D+UDCSeYRn0vufo9Qusa2kTdJOijDMeepOG7Xqhiw from mail.gmail.com>
> Content-Type: text/plain; charset=ISO-8859-1
>
> Does anyone have a protocol for prepping Chlamydomonas for a Scanning
> electron microscope?
>
>
> ------------------------------
>
> _______________________________________________
> Chlamy mailing list
> Chlamy from net.bio.net
> http://www.bio.net/biomail/listinfo/chlamy
>
> End of Chlamy Digest, Vol 75, Issue 2
> *************************************
>



-- 
Heloísa Ciol


More information about the Chlamy mailing list

Send comments to us at biosci-help [At] net.bio.net