[Histonet] General IHC

[Amos Brooks]

Chantel & Julienne,
         Both of these questions are very similar. Likewise for both of you 
I would suggest that you read a publication by DAKO called Basic 
Immunohistochemistry, you can find them at www.dakocytomation.com it is a 
very easy read and is the best crash course that I've seen. It will give 
you an overview that should give you enough information to get you started. 
Lawrence True also has a book (by the same title I think) that is excellent 
as well. (Check with Amazon.com for this)
         You really should know as much about the how and why as possible 
when doing IHC as if anything goes wrong in the process you'll know how to 
fix it. Don't let this intimidate you as most IHC can be as easy or as 
complex as you want it to be. With most automatic stainers you can train a 
monkey to use them. I don't recommend it for the above reasons but it can 
be done. Generally animal IHC is more difficult as diagnostic IHC usually 
is geared toward humans. I mostly work with humans so I can only suggest 
sources of information for animal work. For example I know DAKO has a 
secondary detection kit specifically for animals. There are other vendors 
as well I am sure any of them would love to give you more information.
         Fixation is very important and will radically change the whole 
process. Formalin fixed tissues usually require some step to (for lack of a 
better term) undo the fixation. So, Julienne, you will find there will be a 
difference in the fixed placentas and the frozen ones.
         I suggest that you figure out what antibodies you are going to use 
then determine a vendor from there (usually base this decision on price and 
availability). Then contact a sales rep and ask them to guide you thru the 
process. Most vendors will have very good tech support. (DAKO, Ventana, 
BioCare etc) Some offer classes and tutorials to really train you in how 
things work. DAKO actually has workshops in California that are very good.
Good Luck,
Amos Brooks


At 12:36 PM 10/8/2004, you wrote:
>Message: 10
>Date: Thu, 7 Oct 2004 16:47:41 -0500
>From: "Julienne Rutherford" <jrutherford <@t> sfbr.org>
>Subject: [Histonet] placental IHC for the novice
>To: "Histonet <@t> Lists. Utsouthwestern. Edu"
>         <histonet <@t> lists.utsouthwestern.edu>
>Message-ID: <KCEFILJMHKONHCLECDOIGEDLCAAA.jrutherford <@t> sfbr.org>
>Content-Type: text/plain;       charset="iso-8859-1"
>
>Hello. I'm new to histonet and am not trained in histo or IHC, but am very
>interested. I'm throwing myself into the fray, and hope you can help me. In
>response, which I welcome privately or in the forum, please use simple
>language for I am but a simple woman with a simple dream of studying monkey
>placentas.  I want to study IGF-1 localization in the marmoset monkey
>placenta. I'm doing a lot of reading regarding methods, but I need help as
>to what I need to do (and by extension, how to ask for the money to do it in
>grant applications!). The folks in our histo lab are going to help me and
>train me but I want to come to them more prepared and I have to provide the
>specific materials I'll need. I have both 10% NBF-fixed tissues (most
>sitting around in formalin for a very long time), and I also have frozen,
>but by this I mean stuck in the regular old lab freezer to freeze
>conventionally. I will begin snap-freezing placentae we collect from now on,
>but what can I get out of this old stuff? Can I thaw out what I have and
>snap-freeze them in OCT? Should I try to do IHC on both the formalin-fixed
>AND the frozen tissue?
>
>In order to build a budget I need to know everything I need to make these
>slides. So, what kind of slides and coverslips do I need, what are all the
>chemicals involved, what is a secondary antibody and why do I need it, is a
>chromagen different from a stain, etc.? Also, is there something akin to and
>"IHC for Dummies" resource? I've looked at ihcworld.com for protocols but
>it's over my head at this point. I recognize that this is a skill perfected
>over many years, but I really would like a primer on the basics so I can
>have a more informed discussion about it.
>
>If the elementary pitch of my questions is inappropriate to the forum, I
>sincerely apologize. I'm just not sure where else to turn. Please respond to
>my Texas e-mail if you'd prefer to school (or scold) me privately!
>
>Thanks in advance,
>Julienne Rutherford
>
>Research Assistant (Adjunct)
>Southwest Foundation for Biomedical Research
>Southwest National Primate Research Center
>P.O. Box 760549
>San Antonio, Texas 78245-0549
>phone (210) 258-9864
>fax (210) 258-9883
>jrutherford <@t> icarus.sfbr.org
>
>Ph.D. candidate
>Indiana University
>Bloomington, Indiana
>jnruther <@t> indiana.edu