Ncl l er 6f11

Formalin-fixed paraffin embedded tissue was sectioned at 6 μm. The sections were deparaffinised in xylene and rehydrated through graded alcohol. Sections were either stained with haematoxylin and eosin or processed for immunohistochemistry. H&E stained corneal sections at 72 hours and day 14 post-cautery were assessed by a pathologist, who was masked to the sex of the animal from which the sample was derived. Semi-quantitative scoring of the inflammatory infiltrate present within the cornea (excluding hypopyon) was performed. For immnohistochemical analysis, endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Sections were then incubated with estrogen receptor antibody (1:200, NCL-L-ER-6F11, Leica Biosystems, Wetzlar, Germany), androgen receptor antibody (1:100, N-20 sc-816, Santa Cruz Biotechnology, Dallas, TX, USA) or VEGF-R1 antibody (1:100, ab32152, Abcam, Cambridge, United Kingdom) overnight. Detection was performed using the NovoLink polymer detection system (Novocastra Laboratories, Newcastle Upon Tyne, UK) according to the manufacturer’s instructions. Nuclear labelling was considered specific for androgen and estrogen receptor, while cytoplasmic labelling was considered specific for VEGF-R1.

Whole sections of formalin-fixed, paraffin-embedded tissue were deparaffinised, rehydrated and incubated in 0.5% hydrogen peroxide for 10 min and trypsin for 10 min or 10 mM citrate buffer (pH 6.0) in a pressure cooker for 1 min. Endogenous biotin was blocked with the Dako Cytomation. Sections were incubated with monoclonal antibodies against TFF3 protein, TFF1 protein (Westley et al. 2005 (link)), oestrogen receptor (NCL-L-ER-6F11; Leica Biosystems, Newcastle Upon Tyne, UK) or progesterone receptor (NCL-L-PGR-312; Leica Biosystems), secondary antibody and avidin-biotin immunoperoxidase complex (Vector Laboratories, Peterborough, Cambridgeshire, UK), developed with diaminobenzidine and counterstained with haematoxylin.

After deparaffinization, rehydration, and antigen retrieval in citrate buffer, the slides were incubated for 30 min in PBS with appropriate normal serum at room temperature, which was followed by overnight incubation at 4 °C in a solution of PBS with appropriate normal serum containing a mixture of primary antibodies. After 5 washes (10 min each) in PBS, the specimens were then incubated for 1 h at room temperature with a mixture of secondary antibodies diluted in PBS. Indirect double immunofluorescence for identification of sex steroid hormone receptors in sections was performed with monoclonal mouse estrogen receptor (1:50; NCL-L-ER-6F11, Leica Biosystems), monoclonal mouse progesterone receptor (1:100; Clone PgR636, Dako), monoclonal mouse anti-Inhibin β-A antibody (1:100; sc-166503, Santa Cruz Biotechnology), and secondary goat anti-mouse Alexa 488-conjugated antibody (1:400; 115-545-146, Jackson ImmunoResearch). Appropriate negative and positive controls were performed. Finally, the slides were washed with two changes (10 min each) of PBS, cover-slipped with fluorescence mounting medium (Dako, Denmark), and covered with Menzel-Gläser glasses. The labeled specimens were analyzed immediately.

Ki67% staining (MIB-1 clone code n. M7240, DAKO UK Ltd; working dilution 1:40) was the primary biomarker end point for the POETIC trial and was centrally analysed on all formalin-fixed samples using a single protocol (either core-cut in formalin-fixed, paraffin-embedded or excision specimens in formalin-fixed, paraffin-embedded) as previously described22 (link). All staining was performed on a Dako autostainer using strict adherence to a single staining protocol. Haematoxylin and eosin staining was used to exclude samples with low tumour purity (<40%).
HER2 status was measured locally using immunohistochemistry and/or in situ hybridization24 (link). Biomarker results are shown in Supplementary Table 5.
ER expression of baseline specimens was measured by immunohistochemistry (6F11 clone code n. NCL-L-ER-6F11, Leica Biosystems Ltd; working dilution 1:50) on formalin-fixed samples25 (link). Patients were excluded from this substudy if they were described as ER negative (<1% positive staining of tumour nuclei).
Cellularity was measured by 10 × 10 mm eye-piece graticule with × 40 objective graticule. Nuclei were counted within the grid of at least five fields and the mean values from these measurements were used.
Patients with unsuppressed estradiol upon treatment were excluded.

We performed immunohistochemical studies to investigate immunoreaction of target antigens in the serial section of biopsies using the following antibodies: ERα (estrogen receptor-α, 1:50, NCL-L-ER-6F11, mouse monoclonal, Novocastra Laboratories Ltd, Newcastle, UK); PR (progesterone receptor, 1:40, NCL-L-PGR-1A6, mouse monoclonal, Novocastra Laboratories Ltd, Newcastle, UK); anti-human von Willebrand factor (VWF) antibody (clone F8/86, code M0616; Dako, Denmark, 1:50 dilution), a mouse monoclonal antibody, to investigate immunoreaction to HUVECs. GnRHR (AT2.G7:sc-57176, Santa Cruz Biotechnology, Inc. CA), a mouse monoclonal antibody against both type I and type II receptor (1:25 dilution), was used to immunolocalize GnRHR expression in HUVECs. Non-immune mouse immunoglobulin (Ig) G1 antibody (1:50 dilution, Dako, Denmark) was used as a negative control.

Tumor and metastatic samples were fixed in 4% paraformaldehyde and embedded in paraffin. Antigen retrieval was performed using pH9 antigen retrieval buffer (DAKO S2375) at 95 °C for 20 min or citrate buffer pH6 at 95 °C for 20 min for CC3. Antibodies against ER (NCL-L-ER-6F11, Novocastra), PR (NCL-L-PGR-312, Novocastra), HER2 (SP3, Spring Bioscience), or pan-cytokeratin (DAKO, Clone AE1/AE3) were used at 4 °C overnight, followed by biotinylated anti-IgG secondary antibodies (Vector Labs). The signal was detected by incubation with ABC Elite (Vector Labs) for 30 min and 3,3′-diaminobenzidine (Dako) for 5 min at room temperature.