Ab261736

Immunohistochemistry (IHC) for P-gp was performed on the paraffin-embedded placenta, and FM blocks were prepared as described in section 2.3. Placental tissue served as a positive control, as P-gp is known to be expressed in the placenta (Mathias, Hitti, and Unadkat 2005 (link); Ni and Mao 2011 (link)). FMs without the application of the P-gp primary antibody functioned as the negative control.
Tissue sections (5 μm thickness) were cut and adhered to positively charged slides. After deparaffinizing with xylene, sections were rehydrated with 100, 95, and 70% ethanol. An IHC kit from Abcam (ab64264) was used, and the manufacturer’s instructions were followed for staining. For immunostaining, placenta and FMs sections were incubated with the P-gp primary antibody (Abcam [ab261736]; 1:1000 dilution) at 4°C overnight. For the negative control, FMs sections were incubated overnight in 3% BSA TBS-T without the primary antibody. The IHC antirabbit antibody (1:500, Vector Laboratories, CA, USA) was added, and samples were incubated for 10 minutes at room temperature. Subsequently, DAB as a chromogen and hematoxylin as a counter stain were added for color development. Slides were examined with bright field microscopy for the presence and localization of P-gp; images were taken at 20× and 40× magnification.