FISH Protocol For Paraffin Embedded Tissue Samples

Paraffin-embedded tissue is one of the most widely practiced methods for sample preservation and archiving. It is estimated that, over a billion tissue samples, most of them paraffin-embedded, are being stored in numerous hospitals, tissue banks, and research laboratories in the word. These archived samples could potentially provide a wealth of information in retrospective molecular studies, especilally FISH.

Deparaffinization of paraffin-embedded tissue sections

1)	Paraffin-embedded tissue must be sliced at a thickness of 4-10 μm using a microtome and mounted onto a microscope slide
2)	Prewarm 40 ml 0.01N HCL in a jar in 37°C water bath
3)	Age slide at 90°C for 25 min
4)	Immerse the slide-mounted tissue section in 100% xylene for 15 minutes; repeat in fresh 100% xylene for an additional 15 minutes
5)	Immerse the slide in 100% ethanol for 5 minutes; repeat in fresh 100% ethanol for an additional 5 minutes
6)	Air dry slide
7)	Pre-warm a jar with 10 mM Citric acid to 80°C in water bath
8)	Place slide into Pepsin solution for 30 min
9)	Rinse slide in 70% ethanol 30 second
10)	Air dry slide and check slide for proper digestion; reveal dark distinguishable cells
11)	Dehydrate slide through 70%, 85% and 100% Ethanol each 2 min
12)	Air dry slide and proceed to hybridization

Co-denaturation and hybridization
Prepare probe mix per hybridization for 22 mm² coverslip. Add the probe on the processed tissue slide and place coverslip taking care that there are no air bubbles.

Hybridize the slides with following program:

Denaturation: 83°C for 3 min.
Hybridization: 37°C over night

FISH Protocol For Paraffin Embedded Tissue Samples

Creative Bioarray provides services for entire FISH process with unprecedented analytical accuracy, you can find more at: Histology Services

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