Genomics and Applied Biology 2018, Vol.9, No.9, 56-61
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3 Materials and Methods
3.1 Research materials
Oreochromis niloticus
was purchased in Zhanjiang Seafood Market (about (300 ± 10) g/tail). PBS buffer solution
was purchased from BBI Life Sciences Company of China. RPMI-1640 medium and fetal bovine serum were
purchased from Gibco Company of America. Primary antibody NCCRP-1 Antibody (5C.6) was purchased from
Novus Company of America. Fluorescent-labeled secondary antibody Goat Anti-Mouse IgM-FITC was purchased
from American Southern Biotech Company. HRP-anti-mouse IgM was purchased from Wuhan Boster Biological
Company. Desktop high-speed freezing centrifuge Centrifuge 5810R was purchased from German Eppendorf
Company. Flow cytometry BDFACS Verse flow cytometer was purchased from American BD Company.
3.2 Immunohistochemical analysis
After the fish was anesthetized, 75% alcohol was used to wipe the fish body, and the corresponding tissues were
obtained by dissecting the fish body. After that, the tissues were immediately put into 10% formalin and fixed at
4°C for 24 hours. Then, the tissues were embedded in pre-heated paraffin wax and sliced by a continuous slicing
machine after fixation. During dewaxing, the slices were put into xylene for 15 minutes, and then taken out and
put into new xylene for 15 minutes, after which the slices were taken out and put into anhydrous ethanol for
5 minutes, and then taken out and put into new anhydrous ethanol for 5 minutes. After that, the slices were
continuously replaced and soaked in 85% alcohol and 75% alcohol for 5 minutes, and finally the slices were
cleaned with single distilled water. Next, we put the tissue slices into an antigen repair box and completely
immersed them in the EDTA antigen repair buffer with pH = 8.0. In the case of non-drying slices, we first heated
and boiled the slices in a microwave oven, then stopped heating and kept the temperature for 8 minutes, and later
heated for 7 minutes with a small fire. After cooling at room temperature, the slices were washed 3 times with
pH = 7.4 PBS for 5 minutes each time. The slices were incubated in 3% hydrogen peroxide solution for 25 minutes at
room temperature in dark environment, then the slices were washed 3 times with pH = 7.4 PBS again for 5 minutes
each time to block endogenous peroxidase. PBS on the surface of the slice was gently sucked dry with absorbent
paper. In order to prevent the antibody from flowing away, we coiled the sample tissues with Pap Pen, and added
10% normal rabbit serum in the tissues at room temperature to seal the tissues evenly for 30 minutes. Then, the
blocking liquid was gently thrown away, as well as the primary antibody dissolved in PBS was dripped into the
slices which were incubated overnight at 4°C in a wet box. Next, the slices were washed 3 times with pH = 7.4
PBS for 5 minutes each time. Using absorbent paper to dry the PBS on the surface gently, HRP-labeled secondary
antibody was dropped onto the slices, making ensure that the secondary antibody could cover the tissue evenly
and then the tissue was incubated at room temperature for 50 minutes. Then, the slices were washed 3 times with
pH = 7.4 PBS for 5 minutes each time. We used absorbent paper to dry the PBS on the surface gently, and freshly
prepared DAB coloring solution was dropped onto the slices. The positive stain was claybank. When positive
signals were found under the microscope, the stain was terminated and the slices were washed with distilled water.
We stained the slices with hematoxylin for about 3 minutes, and rinsed them clean with distilled water. We
differentiated them with 1% hydrochloric acid alcohol for several seconds, and then washed them under clean
water. The slices were returned to blue with ammonia water, and then they were rinsed with clean water. The
slices were put into 75% alcohol for 6 minutes, 85% alcohol for 6 minutes, and absolute alcohol for 6 minutes
successively. After that, the slices were soaked for 6 minutes in the new anhydrous ethanol and 5 minutes in
xylene. Then, dehydration treatment was carried out. The slices were taken out, dried and later they were sealed
with neutral gum. Finally, we put the slices under a microscope to observe and take photos.
3.3 Isolation, purification and identification of head-kidney NCC
Oreochromis niloticus
in good condition was selected for anatomy. After dissection, the internal organs of tilapia
were normal in shape and color. A total of 2 g kidney tissues were obtained and placed in 400 mesh sieve, as well
as cut with a small dissecting shear and washed with a rubber-tipped dropper at the same time. The head-kidney
tissues were fragmented and dispersed in 60 mL RPMI-1640 medium. We used two layers of Percoll with volume
fraction of 34% and 51% to separate
Oreochromis niloticus
whole lymphocytes, and two layers of Percoll with
different concentration were added to each centrifugal tube. 0.85% NaCl solution was added to the Percoll
