Proceedings of MatricesFor IITTEP – ICoMaNSEd 2015
ISBN: 978-602-74204-0-3
Biology Page 530
2. Material and Methods 2.1. Research Design
This research is experimental research using factorial randomized block design 4 x 4 with treatment type and time duration of repeated exposure factors as independent variables, and
the number of mice liver cell that expressing TNFα as dependent variable. Categories of treatment factor consist of: negative control NC; fish positive control FiPC; formalin
positive control FoPC; and fish containing formalin FiFo. While categories of time factor consist of: duration of repeated exposure over 0; 2; 14; and 62 days.
2.2. Materials
BALBc mice
M. musculus
as experimental animal, fresh water fish
Oreochromis niloticus
as food model, formaldehyde 37
PA grade
Merck KGgA, Germany, anti- TNFα antibody
BioVision, Inc., USA, staining kit Universal Dako LSAB
®
+ Kit Peroxidase Dako North America, Inc., USA, poly-L-lysine microscope slide Thermo Scientific, Inc., Germany,
alcohol Merck KggA, Germany, xylol, paraffin, phosphate buffered saline PBS, bovine serum albumin BSA, Triton, NaN
3
, entellan new Merck KGgA, Germany, demineralized water, Meyer’s hematoxylin.
2.3. Animal Cultivation, Preparation, and Test Substances Administration
Fourty-eight male BALBc mice 2.5 months old with 15-25 g in weight were maintained in Animal Physiology Laboratory, Faculty of Mathematics and Natural Sciences, University of
Brawijaya, Malang. Mice were housed in polyethylene plastic container, managed in 27±2 C
with 12 hour photoperiod and provided with fodder and water
ad libitum
. All animal procedures conformed to the institutional ethic regulations concerning the protection of
animals Sprengel
et al.
, 2008; Drummond, 2009. The concentration of formaldehyde in fish flesh was setted to 100 ppm mgkg for FiFo
treatment by mixing 10 mL of 1 wv formaldehyde solution diluted from 37 wv formaldehyde PA grade and 1 kg of refined
O. niloticus
meat then adding demineralized water until the final volume is 1 L. The FoPC treatment is made same with FiFo treatment but
without adding refined fish meat. Just the opposite, the FiPC treatment is made by dissolution of refined fish meat without adding formaldehyde solution. For the NC treatment, it contains
only demineralized water as placebo effect for the gavage force-feeding administration. The volume of each substances that expose to each mice was adjusted to the weight of animals. At
the time of the test substance administration reach the specified exposure based on time factor categories the mice were euthanized by cervical dislocation and then dissected to taking the
liver organ.
2.4. Tissue Fixation, Paraffin Block Cutting, and Immunohistochemical Detection of TNFα
