TITLE: Histological staining of fish gonadal tissue
AUTHORS: Jhennifer Gomes Cordeiro, Caio Maximino, Diógenes Henrique de Siqueira-Silva

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol describes the dissection, fixation, and histological processing of gonadal tissue of small and medium-sized fish. It has been used in our lab to describe the microanatomy of the gonads of Amazonian Characidae, as well as of ornamental fish such as zebrafish.</div></div>

[STEPS]
?. [Collection and fixation of gonads]
Anesthetize and euthanize the animals
?. [Collection and fixation of gonads]
Perform a ventro-longitudinal incision to expose the gonads, Bouin's fixative must be immediately applied on the gonads aiming the prefixation. Then, gonads are removed and immediately placed in the fixative
?. [Collection and fixation of gonads]
2Leave gonads overnight in Bouin's fixative for 24h
?. [Collection and fixation of gonads]
After 24h, the fixative solution must be replaced with 70% ethanol, that should be renewed every day until there is no trace of fixative
?. [Dehydration]
Before dehydration the tissue must be sectioned in the desired shape and fit into the molds
?. [Dehydration]
Samples need to be placed in glass vials, to which the solutions will be added
?. [Dehydration]
Transfer the samples to three fast changes of 70%, 80%, and 90% ethanol
[for each change]
?. [Dehydration]
Leave the samples at 95% ethanol for 40 min.
?. [Inclusion]
Include samples in 1:1 solution of 95% alcohol and infiltration resin (Methacrylate glycol) for 40 minutes
?. [Inclusion]
Leave samples in infiltration resin (methacrylate glycol) "overnight" (or at least 12 hours)
?. [Inclusion]
Prepare the inclusion resin solution (Methacrylate glycol) with the hardener (for each 1.0 mL of resin use 0.07 mL of hardener)
?. [Inclusion]
Embedding the material: place the material on the mold and place the resin + hardener solution on it by completing the mold
?. [Inclusion]
After the resin polymerizes, leave embedded samples in a drying stove for 24 h
?. [Inclusion]
Unblock and glue on microtome support stick with epoxy resin glue
?. [Inclusion]
After the glue has completely dried, the sample should be taken to the microtome
?. [Inclusion]
The sample should be sectioned to the thickness of 3 to 5 μm
?. [Inclusion]
The cuts should be deposited on properly identified slides
?. [Inclusion]
The slides should be kept in the drying stove for 1 day and then followed for staining procedures. If staining with H&E, follow steps 19-27. If staining with toluidine blue, follow steps 28-33
?. [Staining: Hematoxylin and eosin]
Moisturize cuts in running tap water for 10 minutes
?. [Staining: Hematoxylin and eosin]
Leave in Harri's hematoxylin solution for 15 minutes
?. [Staining: Hematoxylin and eosin]
1Wash in running tap water for 10 minutes
?. [Staining: Hematoxylin and eosin]
Dehydrate in 90% alcohol with fast change (if eosin is alcoholic)
?. [Staining: Hematoxylin and eosin]
Leave in eosin solution for 15 minutes
?. [Staining: Hematoxylin and eosin]
Rinse well under running water
?. [Staining: Hematoxylin and eosin]
Transfer the samples to three fast changes of 70%, 80%, and 95% ethanol, followed by fast passes in absolute ethanol, and two changes of xylene (3 min for each station) for clearing before coverslipping
[for each change]
?. [Staining: Hematoxylin and eosin]
Transfer the sample to a third change of xylene
?. [Staining: Hematoxylin and eosin]
Mount and coverslip in Permount (xylene-based mounting medium)
?. [Staining: Toluidine blue]
Moisturize cuts in running water for 10 minutes
?. [Staining: Toluidine blue]
Leave in toluidine blue solution for 15 minutes
?. [Staining: Toluidine blue]
Wash in running water for 10 minutes
?. [Staining: Toluidine blue]
Transfer the samples to three fast passes of 70%, 80%, and 95% ethanol, followed by fast passes in absolute ethanol, and two changes of xylene (3 min for each station) for clearing before coverslipping
[for each change]
?. [Staining: Toluidine blue]
Transfer the sample to a third change of xylene
?. [Staining: Toluidine blue]
Mount and coverslip in Permount (xylene-based mounting medium)
?. [Staining: Hematoxylin and eosin]
Check slides on the microscope and take photographs as needed.Example slides: