[Histonet] melanoma tumors and CD markers

From:"Walters, Katherine S"

Histology experts,

I want to harvest and cryopreserve melanoma tumors with adjacent skin
and/or fat tissue and take cut sections at 8-12 mm thickness.  I will
then use immunochemical staining to detect surface markers on various
immune cells including T-cells, B-cells, NK-cells , macrophages and
monocytes.  The surface markers targeted include:  CD3, CD4, CD8, CD19
and CD20, CD16/32, CD49b, CD64 and CD94.  I use a two-step indirect
staining method, and my secondary antibodies are conjugated with Texas
Red.  The melanoma cells express GFP. 

After literature searches, I discovered a rule of thumb unknown to me,
that aldehyde fixation generally inhibits detection of leukocyte surface
markers.  Other researchers reported good results from antigen retrieval
on aldehyde-fixed, paraffin-processed tissue. I hoped for similar
results with my frozen tissue.  

Snap-freeze tissue by placing mold in shallow container of 2-Methyl
Butane chilled with liquid nitrogen.  

The problem I have been experiencing repeatedly is that the melanoma
tumor cells do not maintain good morphological structure during freezing
and/or sectioning.  Because they are fairly large with high cytoplasm
volume and create soft, loose tumors in the mice, I believed that the
cells possibly rupture during freezing or sectioning.  This creates a
problem in that the GFP in the cytoplasm is released upon cell
degradation, and the tumor cells are no longer distinct from other cell
types when viewed by fluorescent microscopy, so staining is messy and
not informative.

Ultimately, I am seeking the method that will provide the best
morphological preservation of the tumor with skin attached, while still
allowing reliable detection of immune cell infiltration using anti-CD
marker antibodies.  If anyone has experience with this methodology or
can make recommendations, it would be extremely helpful to me.  Thank
you for taking the time to read this letter and your assistance is
greatly appreciated!


Jennifer Springsteen
Holden Cancer Research Facility
5210 MERF, University of Iowa
Iowa City, IA  52242

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