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Evaluation of fixation methods for demonstration of N eoparamoeba perurans infection in A tlantic salmon, S almo salar L., gills
Abstract Formaldehyde‐based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross‐linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistoche...
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Published in: | Journal of fish diseases 2013-10, Vol.36 (10), p.831-839 |
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Main Authors: | , , , |
Format: | Article |
Language: | English |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Abstract
Formaldehyde‐based fixatives are generally employed in histopathology despite some significant disadvantages associated with their usage. Formaldehyde fixes tissue by covalently cross‐linking proteins, a process known to mask epitopes which in turn can reduce the intensity of immunohistochemical stains widely used in disease diagnostics. Additionally, formaldehyde fixation greatly limits the ability to recover
DNA
and m
RNA
from fixed specimens to the detriment of further downstream molecular analyses. Amoebic gill disease (
AGD
) has been reliably diagnosed from histological examination of gills although complementary methods such as
in situ
hybridization (
ISH
) and polymerase chain reaction (
PCR
) are required to confirm the presence of
N
eoparamoeba perurans
, the causative agent of
AGD
. As molecular techniques are becoming more prevalent for pathogen identification, there is a need to adapt specimen collection and preservation so that both histology and molecular biology can be used to diagnose the same sample. This study used a general approach to evaluate five different fixatives for Atlantic salmon,
S
almo salar
L
., gills. Neutral‐buffered formalin and seawater Davidson's, formaldehyde‐based fixatives commonly used in fish histopathology, were compared to formalin‐free commercial fixatives
PAX
gene
®
,
H
istoChoice™
MB
* and
RNA
later™. Each fixative was assessed by a suite of analyses used to demonstrate
AGD
including routine histochemical stains, immunohistochemical stains,
ISH
and
DNA
extraction followed by
PCR
. All five fixatives were suitable for histological examination of
A
tlantic salmon gills, with seawater
D
avidson's providing the best quality histopathology results. Of the fixatives evaluated seawater
D
avidson's and
PAX
gene
®
were shown to be the most compatible with molecular biology techniques. They both provided good
DNA
recovery, quantity and integrity, from fixed and embedded specimens. The capacity to preserve tissue and cellular morphology in addition to allowing molecular analyses of the same specimens makes seawater Davidson's and
PAX
gene
®
appear to be the best fixation methods for diagnosis and research on
AGD
in Atlantic salmon gills. |
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ISSN: | 0140-7775 1365-2761 |
DOI: | 10.1111/jfd.12078 |