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Vitreoretinal specimens are extremely difficult to process as a frozen specimen because of their small size and tendency to roll up.1 However, in order to perform immunohistochemical tests it is sometimes necessary to have frozen specimens as antigens may be destroyed if a fixative agent is used.
Dua et al2 and Scott et al3 suggested the use of frozen cucumber as a mount for conjunctival and corneal tissue; we modified this method for vitreoretinal specimens. We describe our technique and provide examples of our results.
Fresh cucumber (obtained from a greengrocer) was cut into small (1 cm3) blocks; the part devoid of seeds was used. We found that with cucumbers older than 2 days the membranes did not adhere sufficiently well. These blocks were then stored at 4°C until required. Pig eyes were obtained and stored at 4°C until required. Subsequently, the eye was placed under the dissecting microscope and basic salt solution injected in through the vitreous cavity to enable easier dissection. The cornea was removed and a vitrectomy performed. The retinal specimens were stained with Indian ink in order to facilitate subsequent localisation.
Following vitrectomy, membranes were removed from humans (these included diabetic membranes, subretinal neovascular membranes, and epiretinal membranes); they were initially placed in Hartmann's solution. Subsequently, they were placed on the cucumber under a dissecting microscope; it was possible to place the membrane flat without it rolling up because of the texture of the cucumber. These membranes were also stained with Indian ink before placing on the cucumber.
The cucumber with the membrane on its “side” surface was placed in an aluminium foil cup and covered with a cryomatrix of Tissue-Tek OCT compound (Fig 1). The foil cup was then put in a plastic container and the contents flash frozen in liquid nitrogen.
The membranes were cut with a cryostat in 4–5 μm sections. We stained one slide from each specimen with haematoxylin and eosin and performed immunohistochemistry on the others (Figs 2 and 3).
No specimens were lost while performing this technique of processing specimens.
We were able to maintain the orientation of the specimens and managed to obtain sufficiently satisfactory specimens to perform our immunohistochemical studies. The use of Indian ink allowed us to locate the specimen easily when cutting sections. Unlike previous studies we found that the specimen attached to the cucumber without the use of albumin.
Ocular tissues that have been processed in this fashion are conjunctiva and cornea.2,7 Whittle et al described a technique using cucumber as a mount for processing cadaveric human retina,8 which enabled indirect immunofluorescence studies.
Retinal specimens are difficult to process because of their size, tendency to roll up and, hence, difficult orientation. Nevertheless, it is necessary to process specimens in this way to perform certain immunohistochemistry techniques.
We suggest that cucumber is a suitable mount for vitreoretinal membranes that are required as frozen specimens for immunohistochemistry. It should be noted that most modern immunohistochemistry may be performed on fixed tissue.
Presented at British Association of Ophthalmic Pathologists Annual Meeting, Dunchurch, March 2002
Each author states that he has no proprietary interest in the development or marketing of any product used in this study.