IHC for BCRP was performed on the paraffin-embedded FM and placenta blocks prepared as above. Placental tissue served as a positive control as BCRP is known to be expressed in the placenta [10 (link)]. FMs without the application of the BCRP primary antibody served as the negative control.
Five-µm tissue sections were cut and adhered to slides with a positive charge. Deparaffinization was performed with Xylene. Sections were rehydrated with 100% ethanol, 95% ethanol, and 70% ethanol. An IHC kit from Abcam (ab64264) was used and the manufacturer’s instructions were followed. An antigen retrieval system was used for epitope unmasking utilizing the Tris-EDTA buffer (pH = 9.0). Placenta and FM sections were incubated with a BCRP primary antibody (Abcam [ab3380]; 1:1000 dilution) at 4 °C overnight for immunostaining. For the negative control, FM sections were incubated overnight in 3% BSA TBS-T without a primary antibody. An IHC antirabbit antibody (1:500, Vector Laboratories, CA, USA) was added for 10 minutes at room temperature, followed by DAB as a chromogen and hematoxylin as a counter stain for color development. Slides were examined with bright field microscopy for the presence and localization of BCRP. Images were taken at 20× and 40× magnification.
Five-µm tissue sections were cut and adhered to slides with a positive charge. Deparaffinization was performed with Xylene. Sections were rehydrated with 100% ethanol, 95% ethanol, and 70% ethanol. An IHC kit from Abcam (ab64264) was used and the manufacturer’s instructions were followed. An antigen retrieval system was used for epitope unmasking utilizing the Tris-EDTA buffer (pH = 9.0). Placenta and FM sections were incubated with a BCRP primary antibody (Abcam [ab3380]; 1:1000 dilution) at 4 °C overnight for immunostaining. For the negative control, FM sections were incubated overnight in 3% BSA TBS-T without a primary antibody. An IHC antirabbit antibody (1:500, Vector Laboratories, CA, USA) was added for 10 minutes at room temperature, followed by DAB as a chromogen and hematoxylin as a counter stain for color development. Slides were examined with bright field microscopy for the presence and localization of BCRP. Images were taken at 20× and 40× magnification.