RE: [Histonet] ISH on fresh frozen tissue

From:Tony Henwood
Margarete,
 
ISH on frozen sections is possible. I have had success with the following to demonstrate mRNA. The nucleolus stains up beautifully:

SOLUTIONS:

1. Acetic Alcohol

Absolute Alcohol 15ml

Acetic Acid 5ml

2. Absolute Alcohol

3. Cold Acetone

METHOD:

  1. Air dry frozen sections (6-8um) minimum 1 hour.
  2. Fix in Acetic Alcohol 15 minutes.
  3. Wash in absolute alcohol.
  4. Place in cold acetone, allow to warm to room temperature.
  5. Remove slides and air dry.
  6. Circle sections with PAP pen, add probe and continue with ISH.
NB do not treat with enzyme or acid.
I would suggest sealing the coverslip with nail polish (after adding th probe) and heating on a hot plate to denature the DNA if searching for DNA sequences.
 

Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

http://www.histosearch.com/homepages/TonyHenwood/default.html
http://us.geocities.com/tonyhenwoodau/index.html

-----Original Message-----
From: Margarete Vermehren [mailto:m.vermehren@anat.vetmed.uni-muenchen.de]
Sent: Tuesday, 30 September 2003 23:57
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] ISH on fresh frozen tissue

I am trying to do ISH on fresh frozen mouse testis. The morphology was o.k. but my protocol doesn’t work. This material should not need pretreatment, I suppose, although I must be wrong somewhere – it doesn’t work that way. Pretreatment with protease E is very delicate. Has anybody got experience he/she could share with me?

Thank you very much!!

Margaret

 

Margarete Vermehren

LMU München / Tieranatomie II

Tel:  +49 89 21805857

Fax: +49 89 21802569

m.vermehren@anat.vetmed.uni-muenchen.de

 



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