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Leica Biosystems Leica HER2 FISH System - 30 Test Instructions for Use TA9217 EN-CE-Rev_D 08/04/2013
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bandpass uorescence microscope lter sets optimized for use with the Leica HER2 FISH
System - 30 Test are available for most microscope models. The recommended lter sets
for the Leica HER2 FISH System - 30 Test are the DAPI/9-Orange dual bandpass, DAPI/
Green dual bandpass, Green/Orange(V.2) dual bandpass and the DAPI/Green/Orange
(V.2) triple bandpass.
C. Methodology
• Prior to undertaking this methodology, users are required to be suitably trained in the
automated in situ uorescence technique.
• Each test section stained with the LSI HER2/CEP17 Dual Probe enables same cell
analysis of both HER2 and centromeric chromosome 17 signals. A subsequent ratio of
HER2 to chromosome 17 signals will enable a quantitative value to be assigned to the
sample, indicating a negative (non-amplied) or positive (amplied) result. Equivocal
(borderline) results (1.8-2.2) should be interpreted with caution. Count an additional 20
nuclei and recalculate the ratio.
D. BOND Enzyme Pretreatment
Prior to staining dilute supplied Leica BOND Enzyme Concentrate 2 at a 1:300 dilution
using supplied Leica BOND Enzyme Diluent in one of the Leica BOND Open Containers
provided. For example, to stain 10 slides prepare 3 mL of working enzyme solution by
diluting 10 µL of Leica BOND Enzyme Concentrate 2 in 2990 µL of Leica BOND Enzyme
Diluent. It is recommended that the enzyme is freshly prepared before each staining run
and that a minimum volume of 900 µL be used per run.
E. Default Staining Protocol
It is recommended that the Leica HER2 FISH System - 30 Test is used with the
recommended default staining protocol shown in Table 2 below.
Protocol Type Protocol Name
Staining *FISH Protocol A
Preparation *Dewax
HIER *HIER 25 min with ER1 (97)
Enzyme *Enzyme 5 for 25 min
Denaturation *D10
Hybridization *ISH Hybridization (12Hr)
Table 2: Default Leica HER2 FISH System - 30 Test Staining Protocol
F. Procedure Steps
These instructions should be read in conjunction with the Leica BOND-MAX and BOND-III
System user manual. A new Leica BOND Universal Covertile should be used with each
slide.
The use of Leica BOND Universal Covertiles, which have previously been utilized for either
immunohistochemical or in situ hybridization staining have not been validated with this test.
1. On the Leica BOND-MAX and BOND-III System, ensure the bulk and hazardous waste
containers have enough capacity to perform the required staining runs.
2. Ensure there is adequate alcohol, distilled or de-ionized water, Leica BOND Dewax
Solution, Leica BOND Epitope Retrieval Solution 1 and Leica BOND Wash Solution
in the bulk reagent containers to perform the required staining runs.
3. Ensure that a clean Leica BOND Mixing Station is installed.
4. Turn on the Leica BOND-MAX and BOND-III System.