SV: RE: [Histonet] Laser Capture Microdissection and thick rat brainsections

From:christina bisgaard



Hi Bilqees,
   
  The reason I work with native rat brain sections is that formaldehyde fixation cross links the proteins in the tissue, resulting in a "false" 2D protein pattern.
  So I guess fixing in 37% formaldehyde is not compatible to my protocol??
   
  Christina



"Bhatti, Bilqees"  skrev:  Hi Christina,

Try to bring brain sections to room temperature first, fix in 37 % formaldehyde for 5-10 minutes. These are thick sections, rinse in distilled water gently for 3-5 minutes. Then proceed with your protocol. Are you using 
75% alcohol for fixation? You can skip this step when you fix in formaldehyde. Good Luck!

Bhatti, B

Baylor College of Medicine 
Center for Comparartive Medicine
Houston, Texas
W # 713-798-6592
U.S.A


-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of christina bisgaard
Sent: Wednesday, October 17, 2007 6:28 AM
To: histonet@lists.utsouthwestern.edu
Subject: [Histonet] Laser Capture Microdissection and thick rat brainsections

Dear Histonetters

I'm in urgent need of help

Does anyone out there have experience with laser capture micro-dissection (Arcturus, Veritas) and thick (60-80) rat brain section?

I have problems collecting more than one Dentate Gyrus (DG) subregion from the hippocampal area from rat brain sections onto the macro cap. I have tried both PEN membrane glass slides and FRAME slides. 

Could this be due to lack of dehydration? When staining/dehydrating 60 horizontal sections, I observe severe horizontal tearing of the sections if left for more than 30 sec in each dehydration step. If put in Xylene, they are totally broken. Even at 30 sec we sometimes see "blackness" of parts of
the sections.

My staining protocol is: (using this protocol we don't see much horizontal tearing)
1) Directly from -80oC to 75% EtOH, 30sec
2) dH2O, 30sec
3) Stain, Toluidin Blue, 3 min.
4) dH2O, 30sec
5) 50% EtOH, 30sec
6) 75% EtOH, 30sec
7) 96% EtOH, 30 sec
8) 100% EtOH, 30sec

I wish to utilize the cap as much as possible (up to 8 DG per cap) in part for economic reasons. Since the downstream process is 2D gels and protein analysis I need as much tissue from each brain as possible which is why I wish to use thick sections to decrease the number of sections to be
microdissected.

I'm kind of stuck right now and running out of ideas. I will appreciate any help and will provide more information concerning our protocol if needed. 

Christina Bisgaard, PhD Stud., Center for Psychiatric Research, Denmark



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