[Histonet] Grad Student Problem

[Terri Braud]

My response:
My first question is what temperature are the blocks that are too brittle? And at what temperature are you trying to cut?  Blocks stored in cryofreezers at -70o C or less are far too cold to cut without brittleness.  My suggestion would be to pull the blocks and put them into the cryostat at -18o C for at least 6 hours to acclimate to that temp, then try to cut and stain for IF.

If that doesn't work, then I would thaw in formalin and process as routine tissue for formalin fixed paraffin embedded.  The process that your student described in the 2nd step is not a complete, or valid process.
Depending on the T-Cell markers they are trying to demonstrate, one can successfully use standard IHC with appropriate clones, or with usually less success, use a modified IF stain procedure for FFPE sections. Since there are a "ba-zillion" T-cell markers, any further details are very dependent on the markers and the condition of the tissue.
Sincerely, Terri Braud

Terri L. Braud, HT(ASCP)
Anatomic Pathology Supervisor
Laboratory
Holy Redeemer Hospital
1648 Huntingdon Pike
Meadowbrook, PA 19046
ph: 215-938-3689
fax: 215-938-3874
Care, Comfort, and Heal
Today's Topics:

   1. grad student problem (Roberta Horner)
Message: 1
Date: Tue, 3 Jan 2017 18:05:15 +0000
From: Roberta Horner <rjr6 at psu.edu>
I got the following from a grad student here at Penn State. I am not sure how to solve his problem if possible. Does anyone have any suggestions I can forward?
Roberta Horner
Animal Diagnostic Lab
Penn State University

"I am having some difficulties sectioning mouse tumor samples for immunofluorescent analysis.  We originally went the OCT route because we are staining for T cell markers and were worried that the heating that occurs during paraffin embedding would compromise the T cell receptor.  The samples are a little old, but we are hoping to section and stain for immune cell infiltrates.  When sectioning with the cryostat, the tissue and OCT is quite brittle and the sample is not intact enough to transfer to a slide. Two colleagues have given the following suggestions:
1. Thaw the OCT blocks, remove the tissue, and place in a new block with fresh OCT media.  I've tried this technique on a few practice samples and the new OCT media seems to be less brittle and I'm able to get tissue on a slide, however the tissue itself still seems to be poor with either freeze or sectioning artifacts.
2. Thaw the OCT blocks in water, remove the tissue and place in formalin overnight, place in 70% EtOH, then paraffin embed.  Section on a microtome.  Check the fluorescently labelled antibody data sheet to see if paraffin embedding interferes with binding.  Try to stain and see what happens.

I was hesitant to try the second suggestion because I have found no protocol that takes tissue originally stored in OCT blocks and subsequently redirects them to formalin and paraffin for microtome sectioning.  If you have any recommendations on how to move forward and section these difficult samples, or know anyone at the diagnostics lab or at Penn State that could help, that would be much appreciated!"

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