[Histonet] Safranin O staining

Victor Wong vhlwong <@t> yahoo.com
Wed Feb 20 01:52:25 CST 2013 

Hi Liz,
 
Get it.  Thank you for the detailed procedures.
 
Best Regards,
Victor

From: Elizabeth Chlipala <liz <@t> premierlab.com>
To: Victor Wong <vhlwong <@t> yahoo.com>; Tony Henwood (SCHN) <tony.henwood <@t> health.nsw.gov.au>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Wednesday, February 20, 2013 12:15 AM
Subject: RE: [Histonet] Safranin O staining


Victor
 
We process these samples on the tissue processor and not manually.  You can use a kimwipe but we prefer the nylon tissue bags (the small ones 30 x 50 mm), they are a bit more expensive but they work well for us.  We receive the pellets in a conical tube, we add a bit of eosin, about a ml, to the formalin in the tube and let it sit for about 10 minutes.  We then use disposable pipets to retrieve the pellet from the conical tube, we don't use forceps at this stage, we draw up the pellet into the conical tube, open the tissue bag and then squirt the solution and pellet into the bag.  Its helpful to have the pellet stained with eosin this way you can actually see it.  We fold the bag and place it into the cassette.  We embed in the pellets in the 15x15 mm base molds.  We capture 4 levels per slide for a total of 8 sections, since the samples are so tiny we collect all the tissue onto unstained slides, this allows us to have sections through the
 pellet.  What we do is collect several ribbions of tissue with about 15-20 sections per ribbon and then pick up 2 sections at a time.  I will attach a diagram from our technical manual to help out in another e-mail.  I'll see if I can get access to the paper.  I can't comment on how to get rid of the debris since we do not perform that process.
 
Liz
 
Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
liz <@t> premierlab.com
 
Ship to address:
 
Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504
From: Victor Wong [vhlwong <@t> yahoo.com]
Sent: Monday, February 18, 2013 8:37 PM
To: Elizabeth Chlipala; Tony Henwood (SCHN); histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Safranin O staining


Hi Liz,
 
Thank you for your response and your invaluable experience in processing such samples.  I'll try manual processing on next batch of samples.
 
I am new to handle these samples.  There would be a lot of cell debris during culture mixing with small pellet and this may be much bigger than the pellet.  May I know how to get rid of it? 
 
Someone suggested to stain the pellet with eosin and place it in folded Kimwipe for processing.  Is routine automatic staining helpful as we don't have a oven to melt paraffin nearby?  We need to take a 30-minute walk to the lab for processing.
 
BTW, I don't have access right to the paper. Anyway, the staining pictures on the website are very nice. 
 
Once, thank you for your help.
 
Best Regards,
Victor

From: Elizabeth Chlipala <liz <@t> premierlab.com>
To: Victor Wong <vhlwong <@t> yahoo.com>; Tony Henwood (SCHN) <tony.henwood <@t> health.nsw.gov.au>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu> 
Sent: Monday, February 18, 2013 11:37 PM
Subject: RE: [Histonet] Safranin O staining

We have processed and stained these types of samples before.  We do not embed the pellets in agarose prior to processing.  We dye them with eosin and place them in a tea bag and process on a very short cycle - 10 minutes per station, embed section and stain.  We have stained with safranin O, alcian blue, toluidine blue and IHC for collagen II.  The safranin O comes out just fine as well as the others. We just receive the samples so I do not know anything about the preparation, collection and fixation.  It possibly could be your construct, we have processed hundreds of these samples and the staining intensity and patterns do change.  I expect this is due to the type of preparation.  If you want to see pictures of the staining, you can go to this website:

http://bioreagents.lifemapsc.com/products/purestem-chondropkg-4d20-8

or this paper:

Sternberg H, Murai JT, Erickson IE, Funk WD, Das S, Wang Q, Snyder E, Chapman KB, Vangsness CT Jr, West MD. 2012 A human embryonic stem cell-derived clonal progenitor cell line with chondrogenic potential and markers of craniofacial mesenchyme. Regen Med. 7(4):481-501

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308
(303) 682-3949 office
(303) 881-0763 cell
(303) 682-9060 fax
liz <@t> premierlab.com

Ship to address:

Premier Laboratory, LLC
1567 Skyway Drive, Unit E
Longmont, CO 80504
________________________________________
From: histonet-bounces <@t> lists.utsouthwestern.edu [histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Victor Wong [vhlwong <@t> yahoo.com]
Sent: Monday, February 18, 2013 12:35 AM
To: Tony Henwood (SCHN); histonet <@t> lists.utsouthwestern.edu
Subject: Re: [Histonet] Safranin O staining

Dear Tony,

Thank you for your prompt reply and the paper.

I do fix the cell before processing in agarose and after embedding in agarose.

It is inevitable to process in wax at higher than 45C as it is set by the routine workers.

It is the first time I worked on cell block precessing.  Do you have protocol of processing chondrogenic pellet by tissue processor?  Any adjustment to my procesing and staining protocol?

I tried to stain the pH1.0 alcian blue for 45 minutes and overnght but unremarkable staining (faint staining) was observed.  Do you think glycosaminoglycans secreted by chondrogenic pellet can be destroyed by heat?
Best Regards,
Victor


From: Tony Henwood (SCHN) <tony.henwood <@t> health.nsw.gov.au>
To: 'Victor Wong' <vhlwong <@t> yahoo.com>; "histonet <@t> lists.utsouthwestern.edu" <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, February 18, 2013 11:45 AM
Subject: RE: [Histonet] Safranin O staining

Victor,

Heat can affect the way tissues and cells bind to dyes. I have attached an article that hints at some of these problems (Allison, R. T. and Bryant, D. (1998)'Effects of Processing at 45 C on Staining', Biotechnic and Histochemistry 73:3,128 -136).

I have also found that some antigens are adversely affected by heat. CEA immunohistochemistry was decreased in cytology cell blocks made with molten agar compared to cell blocks made with fibrin clot. In both cases cell blocks were fixed in NBF after being made. Formalin seemed to protect the antigens in fibrin clots from subsequent heat damage during processing, whereas with agar cell blocks, the heat damage was already done.

Regards
Tony Henwood JP, MSc, BAppSc, GradDipSysAnalys, CT(ASC), FFSc(RCPA)
Laboratory Manager & Senior Scientist
Tel: 612 9845 3306
Fax: 612 9845 3318
the children's hospital at westmead
Cnr Hawkesbury Road and Hainsworth Street, Westmead
Locked Bag 4001, Westmead NSW 2145, AUSTRALIA

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Victor Wong
Sent: Monday, 18 February 2013 1:34 PM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Safranin O staining

Hi all,

I am working on induced chondrogenesis in cell culture.  After, induction, I put the clump of cells (pellets) in 2% agarose to make a cell block.  When the pellet did not sink to the bottom of agarose, I heated to melt the agarose again and centrifuged with higher speed. I fix the agar block in 10% formalin for an hour and then transfer to 70% alcohol and proceeded with normal paraffin procedure.  I stained the paraffin sections with Sigma Safranin O stain but got very faint or no staining.  Here is my protocol:

1. Weigert Iron Hx for 2 min
2. Acid alcohol,Scott Tap and wash
3. 0.02% fast green for 2 min
4. 0.1% acetic acid for 1 min
5. Sigma's Safranin O for 5-10 min
6. 95% alcohol then 3x 100% alcohol @3 min 7. xylene and mount

I also tried to stain the sections with alcian blue pH1.0 and the staining was not quite remarkable.  I'd like to ask if repeated heating on embedded pellet affects staining as I wanted to make sure the pellet was at the bottom of agarose.  Are there other factors affect the staining. Any comments are welcomed.

Thanks you in advance.

Victor
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