The Development of a Frozen Section Technique for Multi-antibody Screening of Impression Cytologies of the Ocular Surface

Abstract

Purpose: : To develop a technique based upon frozen sectioning that permits multi-antibody screening of impressions from the ocular surface using confocal microscopy.

Methods: : A Biopore membrane disc was applied to the temporal bulbar conjunctival surface of human subjects following local anaesthesa with 0.5% proparacaine. Pressure was applied for approximately 10-20 seconds until the membrane became translucent. Upon removal, the membrane was immediately stored at -80 C until further processed. Sections were then fixed in cold acetone (-20°C) for 2 min and dried. This was followed by rinsing in PBS to remove frozen mounting media. Slides were incubated in blocking buffer for 10 min. (0.5%BSAm 1.5% goat serum with PBS) and incubated with antibody diluted in block buffer for 1 hr at RT in a humid chamber. After rinsing in PBS 3 x, the slides were incubated with diluted secondary antibody in block buffer RT for 30 min. After rinsing with PBS slides were mounted with DAPI-1. Antibodies for specific biomarkers were then applied and examined with confocal microscopy.

Results: : Multiple specimens from single impressions of the ocular surface of individual patients could be reproducibly obtained. A variety of antibodies could be applied to the various specimens to allow multiple screening of various biomarkers. Confocal images provided high resolution detail of these layered specimens.

Conclusions: : A frozen section technique has been developed that permits multi-antibody screening of single specimens from the ocular surface using impression cytology. This technique allows maximization of the information obtained while minimizing the need for multiple specimen retrieval.

Keywords: conjunctiva • cornea: tears/tear film/dry eye • cornea: clinical science