Porcine Circovirus type 2 – Systemic disease on pig farms and associated knowledge of key players in the pig industry in Central Uganda

Wilfred, Eneku; Mutebi, Francis; Mwiine, Frank Norbert; James, Okwee-Acai; Lonzy, Ojok

Paired sections were made and stained as described in the IHC protocol provided by the manufacturer (DAKO, Tokyo, Japan). Tissue sections for IHC (spleen, intestines and lymph nodes) were held on silanised slides (DAKO, Tokyo, Japan). Briefly, the tissue sections were deparaffinized in xylene and decreasing concentrations of ethanol before washing in distilled water and Phosphate Buffered Saline (PBS). Endogenous peroxidase was blocked by immersion of the deparaffinized tissues in 3% hydrogen peroxide mixed with 0.5% methanol for 30 min before rising with distilled water and PBS. For antigen retrieval, slides were incubated in freshly prepared trypsin (0.25%) at 37°C for 10 min and then washing with PBS for three times each five minutes. The sections were then incubated at 4°C in PCV2 specific primary antibody (Ingenasa, Madrid, Spain) overnight before rinsing in PBS. This was followed by incubation at room temperature in a secondary antibody (rabbit anti-mouse IgG) conjugated to Horseradish peroxidase enzyme labeled polymer (Sigma, Aldrich, Germany). Development was effected with DAB (3,3′-diaminobenzidine tetrachloride) and hydrogen peroxide in PBS for 3 min before rinsing with PBS followed by distilled water. Mayer's Hematoxylin was used as a counterstaining for 3 min. Eventually the sections were dehydrated in graded alcohol (70%X2 for 3 min each, 100%X2 for 20 min each), cleared with xylene for 20 min before mounting with DPX.