Histoclear histochoice

Tissue histology was used as the secondary tool after PCR, to estimate the severity of, and potential host immune responses to, any haplosporidian infection (e.g. melanisation reactions, haemocyte encapsulation). Three pairs of gills and three portions (ca.0.5 cm³) of the hepatopancreas/gonad were excised and fixed in Davidson's seawater fixative for 24 h prior to their storage in 70% ethanol. Samples were dehydrated in a graded series of ethanol, transferred to Histoclear/Histochoice (Sigma-Aldrich, Dorset, UK) and infiltrated with molten wax using a Shandon™ automated tissue processor (Thermo Fisher Scientific, Altrincham, UK) prior to embedding. Blocks were cut at 5–7 μm thickness using an RM2245 microtome (Leica, Wetzlar, Germany). Sections were mounted on glass slides using glycerine albumin and stained with Cole's haematoxylin and eosin. Stained slides were viewed and imaged using an Olympus BX41 microscope. Images were adjusted for colour balance and contrast only.

Tissue histology took place according to Davies et al. [14 (link)] and was used as the secondary tool after PCR, to estimate the severity of, and potential host immune responses to, any fungal infection. Briefly, gills and portions of the hepatopancreas/gonad were excised and fixed in Davidson’s seawater fixative for 24 h prior to their storage in 70% ethanol. Samples were dehydrated in a graded series of ethanol, transferred to Histoclear/Histochoice (Sigma-Aldrich, Dorset, UK) and infiltrated with molten wax using a Shandon™ automated tissue processor (Thermo Fisher Scientific, Altrincham, UK) prior to embedding. Blocks were cut at ca. 5–7 µm thickness using an RM2245 microtome (Leica, Wetzlar, Germany). Sections were mounted on glass slides using glycerine albumin and stained with Cole’s haematoxylin and eosin. Stained slides were viewed and imaged using an Olympus BX41 microscope. Images were adjusted for colour balance and contrast only.