T@@o provide baseline data on the gill tissue lesions in Huso huso under exposure to direct and water-soluble fraction of diesel oil. In both condition, histopathological analyses exhibited many gill lesions, including epithelial lifting, erythrocyte infiltration, lamellar aneurism, hyperplasia, and lamellar fusion. We report here the results on several abnormalities in the gill structure of the studied fish under direct and water-soluble fraction of diesel oil concentrations which could be used as biomarkers of diesel oil pollution.

Normal method of preserving gills is best done by collecting gills in a very fresh condition. It is always better to collect the fish in a moribund/sacrificed state. Dead fish are much more liable to epithelial erosions due to bacterial digestion of epithelial lining.

As soon as you collect keep the gills (a full gill arch) in 10% buffered formalin for 2-3 h. Then change formalin a couple of times within 24 h. Later shift the samples to 50% propanol or ethanol. Decalcification is often required if you wish to visualize the cartilage association with the gill filaments. weak acids such as nitric, hydrochloric of formic acids can be used (generally 8-10% acid is used at 5-6 times the vol of the sample). 2-3 changes of the decalcifying solution is required in 24-36 h for most gill tissue (probably less in your case as your gills are samll). Then before proceeding to graded dehydration processing and hardening, wax impregnation..wash the gills in distilled water or several changes in 50% propanol. Remaining steps of processing are standard.

I have used the following procedures to keep gill samples for more than four months. Immediately after sample collection (from immediately euthanized fish) keep in Baker's Calcium formalin solution for 24 hours. Then transfer the samples to Gum sucrose solution and keep them at 4 °C. It is recommended to change the Gum sucrose solution every 15 days until the analysis. For both cases, make sure your sample perfectly immersed inside the solution. Here are the formula details-

1. Bakers Calcium formalin

40% Formalin=100 ml

Calcium acetate=20 g

Dissolve this and makeup to 1 liter with distilled water.

2. Gum sucrose

Sucrose=30 g

Gum arabic=1 g

Dissolve these into 100 ml of distilled water.

Hope this will help you.