Application suite advanced fluorescence version

Immunofluorescence was performed as described previously [27 ]. Formalin fixed ovarian tissue was embedded in paraffin and 5-micrometer thick sections were cut and mounted on SuperFrost Plus microscope slides. Followed by deparaffinization, slides were rehydrated by running them through xylene and graded ethanol solutions Antigen retrieval was performed by using 0.9% Antigen unmasking solution (Vector Laboratories) and pressure cooked at 20 psi for 5 min. Slides were allowed to cool and sections were blocked with 5% normal horse serum. Sections were incubated with anti-estrogen receptor antibody overnight at 4° C. After washing with 1X PBS with 0.01% Tween 20, sections probed with anti-estrogen receptor antibody (sc-543) were incubated with Alexa 488 conjugated anti-mouse IgG (Jackson laboratories) for an hour at room temperature, washed with 1X PBS with 0.01% Tween 20 and mounted using DAPI Fluoromount G (Southern Biotech). Control sections were incubated with ER alpha antibody pre-absorbed with the ER alpha blocking peptide. Sections were visualized using a Leica DM5500Q microscope and images were captured using a Leica DFC365 FX camera. Images taken from the A4 (DAPI) and L5 (Alexa 488) channels were superimposed using the Leica Application Suite-Advanced fluorescence version 2.6.0.7266 software.

Tissues were collected, fixed and embedded as described in [34 ]. Five micrometer thick sections were cut and mounted on SuperFrost Plus microscope slides. Following deparaffinization, slides were rehydrated through xylene and graded ethanol solutions. Antigen retrieval was performed by using 0.9% Antigen unmasking solution (Vector Laboratories) and pressure cooked at 20 psi for 5 min. Slides were allowed to cool and sections were blocked with 5% normal horse serum. Sections were incubated with anti-estrogen receptor antibody overnight at 4° C. After washing with 1X PBS with 0.01% Tween 20, sections probed with anti-estrogen receptor antibody (sc-543) were incubated with Alexa 488 conjugated anti-mouse IgG (Jackson laboratories) for an hour at room temperature, washed with 1X PBS with 0.01% Tween 20 and mounted using Dapi Fluoromount G (Southern Biotech). Sections were visualized using a Leica DM5500Q microscope and images were captured using a Leica DFC365 FX camera. Images taken from the A4 (Dapi) and L5 (Alexa 488) channels were superimposed using the Leica Application Suite-Advanced fluorescence version 2.6.0.7266 software.