[Histonet] re: plastic embedding

[JR R]

It's been a while, but I used to do plastic sections with a compound called Immuno-Bed. 

First we dehydrated tissue through an acetone series.  From 100% acetone, tissue was then infiltrated in uncatalyzed Immuno-Bed, for...about an hour for tiny samples....then the tissue was finally transferred to plastic embedding molds and covered with catalyzed immunobed.    Heat is released, so it might be a good idea to allow the catalytic hardening process to go overnight at 4 degrees C.  The plastic blocks popped out of the flexible plastic molds pretty easily.

For sectioning, we prepared our own glass knives--You start with a glass rectangle, cut that into squares, then cut the squares into triangles with very, very sharp edges.  As I recall, the diamond-edged cutter used to produce the knives cost about $5,000.

The plastic blocks are cut using a special microtome with a chuck just for glass knives.  It took some practice, but I was able to produce 1 micron sections pretty easily.  The sections DO NOT ribbon.

Seems like I used a dumont forceps to apply the sections to a drop of water on a slide, then drained the water from underneath the section with a tissue.  Or maybe I just let them dry overnight.  Can't remember for sure.

The next day, slides with plastic sections were washed three times in acetone, then rehydrated with water, then etched in a NaOH bath for ...hmmm, maybe 10 minutes, rinsed, soaked in PBS, and then put through a standard immunocytochemistry protocol.


Sorry about fuzziness of details, but it's been about 17 years since I last did the procedure.

Jerry Ricks
Research Scientist
University of Washington
Department of Pathology

> Date: Thu, 29 Jan 2009 14:03:57 -0500
> From: kevinpe <@t> mail.med.upenn.edu
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] re: plastic embedding
> 
> I am in a similar situation as Armando. My PI recently told me to figure out how to do plastic embedding to view calcein/Xylenol orange staining. I've been following a procedure described in " Immunohistochemistry of matrix markers in Technovit 9100 New-embedded undecalcified bone sections" - Eur Cell Mater. 2003 Dec 31;6:57-71; discussion 71 . But I have yet to actually begin infiltrating the bones. If anyone can offer any knowledge and advice I would greatly appreciate it. 
> 
> Thanks, 
> Kevin 
> 
> Kevin Egan 
> Research Specialist 
> Department of Medicine 
> Division of Geriatric Medicine 
> University of Pennsylvania School of Medicine 
> Biomedical Research Building (BRB) II/III, Room 745 
> 421 Curie Boulevard 
> Philadelphia, PA 19104-6160 
> Telephone: 215-573-4005 
> 
> Message: 1 
> Date: Wed, 28 Jan 2009 17:30:47 -0500 
> From: "Armando Tellez" <atellez <@t> crf.org> 
> Subject: [Histonet] Plastic embedding 
> To: <histonet <@t> lists.utsouthwestern.edu> 
> Message-ID: <D48BEC1869EA534288B5F670DFA6E97301CA07B8 <@t> be018.mail.lan> 
> Content-Type: text/plain; charset="us-ascii" 
> 
> Hi, 
> 
> 
> 
> Does anyone have a protocol for plastic embedding that shows in detail 
> the procedure and equipment needed for this purpose that does not mind 
> share it? My office email is attached to my signature. Please let me 
> know since my boss want to start a stent pathology protocls and he 
> claims that we have the equipment, so first I need a good full blown 
> descriptive protocol and all the protocols in internet are already 
> assuming that you have some plastic embedding knowledge and I have never 
> embed any metal devices. (I need a protocol for microtome, not grinding) 
> 
> 
> 
> Thanks 
> 
> 
> 
> Armando Tellez 
> 
> atellez <@t> crf.org <mailto:atellez <@t> crf.org> 
> 
> Pathology Department 
> 
> Tel: (845) 290 8034 
> 
> Fax: (845) 290 8030 
> 
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