Polymer novolink kit

The expression of NRF2 proteins in collected rat brain tissues was assayed using a routine immunohistochemistry assay. In brief, the collected rat brain tissues were prepared into 5 μm thick sections, fixed in paraformaldehyde, embedded in paraffin, de-paraffinized by using xylene, re-hydrated by using gradient ethanol, subjected to antigen retrieval by boiling in a pH 6.0 citrate buffer for 1 hr at 96°C, and then treated by utilizing a Polymer novolink kit (Leica, Nanterre, France) according to the general protocol provided by the manufacturer. After being incubated at 4°C overnight by using anti-NRF2 primary antibody (1:8000 dilution in PBS, Abcam, Cambridge, MA) as well as corresponding secondary antibodies, the slides were counter stained using a DAB substrate and the signal of positive NRF2 expression was detected by using a microscope (IM 1000, Leica, Nanterre, France).

Lung tissue sections 4 μm thick were deparaffinized in xylene and rehydrated through graded ethanol concentrations. After antigen retrieval in citrate buffer at pH 6.0 at 96°C, slides were processed by using the Polymer novolink kit (Leica, Nanterre, France). Slides were incubated overnight at 4°C with polyclonal anti-E2F3 antibody (1:800 in phosphate buffered saline-1% bovine serum albumin-0.1% Triton) and signals were developed with DAB used as a substrate. Signals were analyzed by using ImageJ to quantify the staining within the alveoli under a microscope equipped with a DC 300F camera (digital module R, IM 1000; Leica).