Immunostaining of miR-214 Target Genes

To investigate if silencing miR-214 affected the protein expression of its target genes, immunostaining was performed. Another well-known transcription regulator, transforming growth factor beta 1 (TGFβ 1) is highly expressed in calcified AVs and is known to mediate phenotypic switch (fibroblast to myofibroblast), fibrosis, apoptosis, inflammation and calcification. So the possible regulation and activation of TGFβ 1 by miR-214 was also tested. Frozen sections were fixed in a 1:1 mixture of methanol/acetone for 10 min at room temperature (RT) and then blocked (1 h, at RT) using 10% (v/v) donkey serum in PBS. Immunofluorescence staining was carried out using following antibodies, KLF4 (Novus Biologicals, CO), TGFβ 1 at a concentration of (1: 200) overnight at 4 °C. Secondary staining was performed using appropriate secondary antibody labeled with Rhodamine Red X (Invitrogen, NY) for KLF4 or DyLight 488-conjugated donkey anti-rabbit secondary antibody for TGF β 1 (Jackson Immunoresearch, PA) (1:500). Nuclei were counterstained with DAPI (1: 5000) and the samples were mounted using anti-fade mounting medium and imaged. ImageJ software was used to quantify the expression of the proteins in the immunopositive cells as reported by Thayer et al.54 (link).