Raw Data TNF-alpha & E-selectin immunohistochemical vs ELISA

White rats were euthanized 14 days after being injected with a subcutaneous dose of 0.20 mg equivalent to 2 x 1011 CFU of Bacillus anthracis spores by ketamine injection. Lung tissue was taken and underwent tissue preparations. Each sample was made as a histological slide for immunohistochemical examination conducted at the Anatomic Pathology Laboratory, Faculty of Medicine, Universitas Sebelas Maret Surakarta. A complete necropsy was performed on each mouse. Lung tissues were fixed by submersion in 10% neutral buffered formalin, then embedded in paraffin, stained with H&E, and be evaluated by a board-certified veterinary pathologist. An immunohistochemistry stain was then performed on lung tissues.

Antibodies against murine TNF-α dan E-selectin for mice (Santa Cruz Biotechnology Inc.,   California, USA) were used, dissolved in TRIS-PBS 1:200. Lung tissues were incubated with biotinilyzed antimurine antibodies (Dako Kit) and streptavidin-biotin peroxidase (Dako Kit) for 30 mins. Lung tissues are then washed with PBS pH 7,4, two times, each for 3 mins between tissues incubation before.

At the end of the study, serum collection for TNF-α and E-selectin levels were selected, performed, and analyzed by Enzyme-Linked Immunosorbent Assay (ELISA). 100 µl of serum were added into a sterile test tube, and were incubated for two and a half hours at 4°C to the prepared reagents and samples. Then, a 100 µl biotin antibody, streptavidin, and TMB One-Step Substrate Reagent were added into each tube. These tubes were then further incubated for 1 hour, 45 minutes, and 30 minutes, respectively. The reading was done at 450 nm, and the ELISA Kits that were used for this research were RayBio® Rat, TNF-α, ELISA Kit, dan RayBio®, Rat E-selectin, and Norcross Georgia, USA.