[Histonet] RE: Request to help for rat eye histology - long response

[Elizabeth Chlipala]

We process rat eyes all of the time.  I know that the collection of the eyes is important and I can not comment on that since we do not collect the eyes, but anytime you are handling both mouse or rat eyes for retinal work you need to be as gentle as possible.  For retinal work you need to fix in davidsons fixative (don't know how long, since we receive the eyes in 70% alcohol) formalin fixative will not work the retina will detach.  One we receive the eyes which have been marked with ink as to the superior portion, we trim about 1mm or so on the nasal side (you will also need to know if the eye is right or left to help you determine which is the nasal side).  I trim with a microtome blade and using a small round trimming matrix that the eye sits on top.  The eyes are processed on a same day processing cycle - 20 minutes per station with proper instead of xylene.  We do not process overnight.

70% - 20 minutes
80% - 20 minutes
95% - 20 minutes
100% - 20 minutes
100% - 20 minutes
Propar - 20 minutes
Propar - 20 minutes
Propar - 20 minutes
Paraffin - 20 minutes
Paraffin - 20 minutes
Paraffin - 20 minutes

When embedding we have a deep base mold filled with melted paraffin that we drop the eye into to make sure that there are no air bubbles in the eye caviety.  You can gently move the lens to get the air bubbles out if necessary, but you really do not want to manipulate the eye much since you might cause separation and tearing of the structures. Once we know that the eye has no air bubbles we place it in the appropriate base mold, we use the small square molds with the superior side of the eye towards the cassette label.  We trim into the correct area, we have a microscope next to the microtome so we can make sure that we are at the optic nerve head and then place the trimmed blocks on ice for about 30 minutes or so, we then cut ribbons of 5 micron sections depending upon the study design and place on a good plus slide, let dry overnight and stain the following day with H&E.  Since you are in Denver we are just in Longmont so if you want to stop by and see how we do this just e-mail me back.

Good Luck

Liz

Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC
Manager
Premier Laboratory, LLC
PO Box 18592
Boulder, CO 80308-1592
(303) 682-3949 office
(303) 682-9060 fax
(303) 881-0763 cell
www.premierlab.com

Ship to address:

1567 Skyway Drive, Unit E
Longmont, CO 80504

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Gupta, Himanshu
Sent: Thursday, July 07, 2011 11:38 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Request to help for rat eye histology

Hi

I need an advice from all experts there on histology of Rat eye.

I need to take clear section of eye (cross section) so that I can see each layer of cells clearly. Please advise me step by step process to prepare the histology slides/sections of rat eye.

I try it many times but I am not able to get the clear sections, the cells/layers got broken.

Regards

himanshu

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