Chromogenic in situ hybridization technique for detecting porcine circovirus 3 in lung and lymphoid tissues

Figures

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  Figure-1. Verification of in situ hybridization (ISH) digoxigenin (DIG) probe development by agarose electrophoresis. DIG-labeled probes were successfully developed using porcine circovirus 3 (PCV3) positive samples L43 and L53 as templates. Unlabeled probes were expected to be 69 bp, while corresponding labeled probes were expected to appear larger. The generated probes were used in subsequent ISH staining protocol. Lane 0: DNA ladder (Qiagen® GelPilot 50 bp ladder). Lane 1: Positive band for unlabeled experimental PCV3 probe from sample L43. Lane 2: Positive band for DIG-labeled experimental PCV3 probe from sample L43. Lane 3: Negative control. Lane 4: Positive band for unlabeled experimental PCV3 probe from sample L53. Lane 5: Positive band for DIG-labeled experimental PCV3 probe for sample L53. Lane 6: Negative control. Lane 7: DNA ladder (Qiagen® GelPilot 100 bp plus ladder).

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  Figure-2. (a) Positive in situ hybridization (ISH) results from lung tissues. Multifocal ISH positive signals were observed as brown grains of chromogenic staining within cytoplasm of pneumocytes. 200× magnification with a scale bar equivalent to 100 µm. (b) Positive ISH results from lung tissues. Multifocal ISH positive signals were observed as brown grains of chromogenic staining within cytoplasm of pneumocytes. 400× magnification with scale bar equivalent to 50 µm. (c) Positive ISH results from inguinal lymph node tissue. Multifocal ISH positive signals were observed as brown grains of chromogenic staining within cytoplasm of lymphocytes. 200× magnification with scale bar equivalent to 100 µm. (d) Positive ISH results from mesenteric lymph node tissues. Multifocal ISH positive signals were observed as brown grains of chromogenic staining within cytoplasm of lymphocytes. 400× magnification with scale bar equivalent to 50 µm.

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  Figure-3. (a) Negative in situ hybridization (ISH) results from lung tissues. In situ hybridization positive signals were not observed. 200× magnification with scale bar equivalent to 100 µm. (b) Negative ISH results from inguinal lymph node tissues. In situ hybridization positive signals were not observed. 400× magnification with scale bar equivalent to 50 µm.