Permafluor

Tissue sections were subjected to antigen retrieval in citrate buffer pH6 and processed according to standard laboratory protocols. Sections were first incubated with mouse monoclonal ERβ5 (clone 5/25. BioRad, cat no. MCA4676T) diluted 1:200 in normal goat serum (NGS) overnight at 4°C, followed by goat anti-mouse peroxidase fab (Abcam) 1:500 in serum for 30 min at room temperature and finally incubated with Tyramide Fluorescein (PerkinElmer) at 1:50 in kit diluent for 10 min. Antibody elution was carried out by boiling sections in citrate buffer for 2.5 min followed by 30 min rest, incubated in NGS for 30 min at RT, blocked by streptavidin/biotin following manufacturer’s instructions (Vector, Peterborough, UK). Sections were washed and incubated with ERα mouse monoclonal (Vector, cat no. VP-E614) at 1:80 in NGS overnight at 4°C. Slides were incubated with goat anti-mouse biotinylated (Abcam) at 1:500 in serum for 30 min at RT, followed by Streptavidin Alexa fluor 546 (Molecular Probes) 1:200 in PBS for 1 h. Sections were washed, counterstained with DAPI (Sigma) at 1:1000 in PBS for 10 min before finally mounting in Permafluor (PerkinElmer). All washes between antibodies were carried out three times in TBS. Full details of antibodies used in the study are provided in Supplementary Table 2.

Total tissue ¹⁴C-activity was determined using a Packard System 387 Automated Sample Preparation Unit (Packard Instrument Co., Inc., Meriden, Connecticut, USA), which completely oxidizes the tissue sample into ¹⁴CO₂ which is quantitatively trapped by Carbo-Sorb® (PerkinElmer) in scintillation vials. Following addition of liquid scintillant (Permafluor, PerkinElmer) and mixing, the samples were ready for counting.

Blood samples (duplicate weighed aliquots) were combusted and analyzed by LSC. The remaining blood was centrifuged at ∼2,400 rpm (1,300 g) for ∼10 min at ∼5°C, and the resulting plasma (duplicate weighed aliquots) analyzed by LSC. Urine and cage rinse samples were analyzed directly by LSC. A 50:50 mixture of ethanol and water was added to feces to facilitate homogenization. The sample was then combusted and analyzed by LSC. Sample combustions were done in a Model 307 Sample Oxidizer (PerkinElmer, Inc., Waltham, MA) and the resulting ^{14 (link)}CO₂ was trapped in a mixture of PermaFluor™ and Carbo-Sorb^®. Sample radioactivity was measured in Model 2900TR liquid scintillation counters (PerkinElmer, Inc.) for ≥5 min or 100,000 counts. Each sample was homogenized before radioanalysis. All samples were analyzed in duplicate if sample size allowed. If results from sample duplicates (calculated as ^{14 (link)}C dpm/g sample) differed by >10% from the mean value, the sample was rehomogenized and reanalyzed. Profiling and identification of metabolites versus unchanged lifitegrast and its metabolites was done by HPLC (Xterra MS C18 column; Waters Corporation) followed by liquid chromatography mass spectrometry (LC-MS) and LC-MS/MS (LTQ Orbitrap XL™ mass spectrometer with electrospray ionization [Thermo Fisher Scientific, Waltham, MA]; 610TR radiochemical detector [PerkinElmer, Inc.]).