Looking for Ferns: Optimization of Digestion Pretreatment in Fluorescence In Situ Hybridization (FISH) Technique on Paraffin-embedded Tissues

Abstract

Fluorescence in situ hybridization (FISH) is a useful cytogenetic technique for the detection of chromosome aberrations. However, applying this technique routinely on paraffin-embedded tissue is hampered by technical problems. The efficiency of hybridization is influenced by formalin fixation time, and this may vary considerably between specimens. We present a simple method for improving hybridization by microscopically monitoring the time of enzymatic digestion. To establish optimal digestion time, enzymatic digestion was stopped at 3-minute intervals for biopsies and 10-minute intervals for autopsies in 24 paraffin-embedded samples. At every stop, tissue morphology was examined under light microscopy to determine if observed changes could be correlated with subsequent FISH results. The appearance of fernlike formations was found to mark the optimal digestion time that produced the strongest hybridization signals. Using this method of digestion time control, an additional 41 cases were evaluated for FISH with various types of probe. Monitoring under the microscope could be more spaced if the morphology did not change after the first visual control and could be adapted to the type of sample (in general, endoscopic samples, total digestion time of about 10 min; routine biopsies, 15 to 30 min; autopsy samples, 20 to 40 min). In every case, the appearance of the fernlike pattern correlated with proper hybridization signal. Monitoring digestion time for the appearance of fernlike structures is a useful method for improving reproducibility of FISH technique on paraffin-embedded samples. It is particularly useful when dealing with samples under heterogeneous fixation conditions (consultations, autopsies, etc.), because it eliminates the need for repetition.

Optimization of trypsin treatment condition utilizing immunohistochemistry for chromogenic in situ hybridization

“…Many researchers have developed different protocols to improve the signal detection efficacy of FISH using FFPE tissue sections . These authors have stressed the importance of heat treatment and/or protease treatment.…”

“…Many researchers have developed different protocols to improve the signal detection efficacy of FISH using FFPE tissue sections. [9][10][11][12]14 These authors have stressed the importance of heat treatment and/or protease treatment. Some studies described the methods to optimize protease treatment for FISH by observing the degree of tissue digestion under a microscope before applying the probes, but the evaluation of the degree of tissue digestion may differ among observers.…”

“…Some studies described the methods to optimize protease treatment for FISH by observing the degree of tissue digestion under a microscope before applying the probes, but the evaluation of the degree of tissue digestion may differ among observers. 9,14 Pepsin and proteinase K are the most widely used proteases for FISH and CISH. However, these treatments often cause varying degrees of nuclear and cytoplasmic digestion depending on the samples.…”

“…In contrast to heat treatment, the optimal condition of protease treatment may vary depending on the tissue type and fixation condition. Thus, we need to empirically optimize the condition of protease treatment for each sample to detect successful signals …”

“…Thus, we need to empirically optimize the condition of protease treatment for each sample to detect successful signals. 1,12,14 Trypsin is a pancreatic serine protease with a high substrate specificity that cleaves peptides on the C-terminal side of lysine and arginine amino acid residues. 15 Histone proteins bind nuclear DNA and contain a high proportion (20-24%) of positively charged amino acids (lysine and arginine).…”