The optical/thermochromic behavior has been evaluated in an optical spectrophotometer (Shimadzu UV-3101PC) with an embedded sample heating-cooling cell.
For one case, for which DNA was extracted from the paraffin embedded sample, a focal lack of hMLH1 staining was discovered when IHC was performed on the same tissue sample and on other additional samples.
The ion beam mills through the embedded sample material and uncovers a new surface of the sample which can be imaged with the SEM.
Nevertheless, this event was more frequent when DNA was extracted from a frozen sample that was different from the paraffin embedded sample used for IHC.
From each paraffin embedded sample, adjacent slides of 5 µm sections were stained with various antibodies.
Fresh samples were cut with a hand microtome to 50 µm, embedded samples with a Reichert Ultramicrotome to semi-thin 2 µm and ultra-thin 90 nm TEM sections.
The process of fixation and embedding itself [2] [5] as well as storage of the embedded samples over an extended time period [6] clearly have a decidedly negative impact on the quality of DNA and RNA that can be isolated from such samples.
Genomic DNA was isolated from frozen tissues or from paraffin embedded samples using a standard procedure [6], or the FFPE tissue kit (56404, Qiagen, Hilden, Germany), respectively.
Sections 6 μm thick were cut from the paraffin embedded samples with a microtome (Leica TP 1020 Automatic Tissue Processor) and placed on Superfrost plus slides (Menzel-Gläser, Denmark).
One hundred and sixty three fixed formalin, paraffin embedded samples with a histopathological diagnosis of CIN, cervicitis or cancer in cervical loop excisions and cone specimens were selected from the archives of the Department of Pathology of the Fernandes Figueira Institute (IFF), Fiocruz, Rio de Janeiro, Brazil.
