[Histonet] RE: Immunofluorescence on FFPE skin

[Sue Hunter]

Hi Erin
We do direct immunofluorescence staining on FFPE kidney biopsies but instead of microwave antigen retrieval, we use proteinase K  (20mg/ml  Qiagen Cat # 19131) diluted 1/10 in Tris buffered saline  for 20 minutes at Room Temp.  We also incubate with the antibody (1/5 dilution) for 90 minutes. Don't remember if our student tried using the microwave (it was a student project) but I would certainly give it a try.  There really is no difference with the wattage for a microwave - the trick is to get the solution boiling - THEN start your timing for retrieval.  We do our AR for our FISH testing for 20 minutes at half power after it comes to a boil.  This allows the solution to keep at near boiling temp but not to over flow the container when it comes back to a boil.  The timing for using a pressure cooker would have to be validated - now you are adding an additional parameter of pressure to factor in to bring the solution to a boil. 
Good luck - hope it works for you.
Sue

Sue Hunter, Supervisor
Advanced Diagnostics
Beaumont Health System
Royal Oak MI
248-898-5146
shunter <@t> beaumont.edu


-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Martin, Erin
Sent: Monday, March 18, 2013 2:28 PM
To: histonet
Subject: [Histonet] Immunofluorescence on FFPE skin

Hello all,



My pathologist gave me a copy of "Immunofluorescence with Dual Microwave Retrieval of Paraffin-Embedded Sections in the Assessment of Human Renal Biopsy Specimens" from the American Journal of Clinical Pathology.  He said that the same principle should work on skin and he would like to be able to do IF on fixed tissue in addition to our usual cryostat sections.  Has anyone else read the paper who might be willing to give me some basic advice to try working it out?  Is a microwave necessary (paper's method uses 2 different wattage settings) or is there a way to use HIER in waterbath or pressure cooker?



Thanks in advance,

Erin



Erin Martin, Histology Supervisor
UCSF  Dermatopathology Service
415-353-7248

Confidentiality Notice
The information transmitted is intended only for the person or entity to which it is addressed and may contain confidential and/or priviledged material.  Any review, retransmission, dissemination or other use of, or taking of any action in reliance upon, this information by persons or entities other than the intended recipient is prohibited.  If you receive this in error, please contact the sender and delete the material from any computer.

_______________________________________________
Histonet mailing list
Histonet <@t> lists.utsouthwestern.edu
http://lists.utsouthwestern.edu/mailman/listinfo/histonet