人間の強膜の共焦点顕微鏡法を実行するために積層技術を用いて、

The overall goal of this methodology, is to perform confocal microscopy of the human sclera. This method can help to answer key questions in the ophthalmic field. Such as immune cell distribution and vascular status in the healthy, and the pathological conditions in the Sclera.

The main advantage of this technique is that it allows for the confocal imaging of dense connective tissue. Demonstrating the procedure will be Stefan Kremers, a grad student from our laboratory. To begin, prepare 96 percent Ethanol and PBS in individual tubes.

Then prepare 100 micro-liters per sample of the primary antibodies in the recommended dilution of PBS containing two percent BSA, and keep all solutions on ice. Arrange clean instruments, including one to two Colibri forceps, straight micro-dissecting scissors, curved scissors, a number 10 scalpel, or ophthalmic scalpel micro feather, four 26 gauge needles, and a swab. Wrap aluminum foil around a polystyrene plate, or prepare a cork plate to affix the tissue.

After producing an anteriorly opened bulb according to the text protocol, place the Sclera on a swab with the open part facing up. Then use colibri forceps to remove the retina, and the pigmented uveal layers from the inner sclera until the sclera is free of these layers. Next use curved scissors if necessary to remove the retina and uvea from the papilla.

Then remove the remaining conjunctiva, extra-ovular muscles, and tenons capsule from the superficial sclera. Now using colibri forceps to gently hold the tissue, with straight scissors cut approximately two square centimeter sized scleral samples from different locations on the tissue, avoiding repeated grabbing with the forceps which will make the area unsuitable for confocal microscopy. Place the samples in 1.5 milliliter tubes, add 1.5 milliliters of 96 percent ethanol, and incubate for 15 minutes to fix the tissue.

Then after removing the ethanol, use 1.5 milliliters of PBS to wash the samples three times for five minutes each while shaking. Next, transfer the samples to 1.5 milliliters of PBS with five percent BSA, and incubate at room temperature for two hours which will induce swelling of the samples, to help with laminating and prevent nonspecific binding. When the samples have swelled, under a stereo-binocular microscope use 26 gauge needles that have been bent at the edges to affix the posterior scleral samples to the foil wrapped polystyrene membrane, or cork plate.

Then to laminate the full thickness samples use colibri forceps to horizontally hold the anterior edge of the scleral tissue. With a number 10 scalpel, carefully cut as thin a layer as possible of the superficial sclera, from the underlying layer. To prevent the tissue from drying out pipette 50 micro-liters of PBS onto the layers.

Then after the layers have been cut place them in 100 micro-liters of PBS in a 96 well plate, and use a waterproof marker to label both the orientation and the layer. Continue to isolate layers and transfer to the 96 well plate until the tissue is fully laminated. To carry out Immunohistochemistry, remove the medium from the 96 well plate by holding the pipette against the wall of each well and aspirating the fluid.

Then add 100 micro-liters of the primary antibodies diluted one to 100 in PBS with two percent BSA to the appropriate wells, and incubate at four degrees Celsius overnight. The next day use 200 to 300 micro-liters of PBS to wash the samples on a shaker three times for five minutes each. If needed, use another primary antibody and incubate again overnight at four degrees Celsius.

Then add 100 micro-liters of the corresponding secondary antibodies, diluted one to 300 in PBS with two percent goat serum, and incubate in the dark at room temperature for one to two hours. After the incubation, use 200 to 300 micro-liters of PBS to rinse the samples again on the shaker three times for five minutes each. Then add 100 micro-liters of a one to 2000 dilution of DAPI to each well and incubate in the dark at room temperature.

Before using 200 to 300 micro-liters of PBS to wash the samples two times. Transfer the samples onto microscope slides under the stereo-binocular microscope to make sure the sample is lying flat on the slide, and add one to two drops of fluorescent mounting medium. Then place a cover slip on top and use transparent nail polish to seal the edges.

Store in the dark at four degrees Celsius for later use or examine directly by confocal microscopy. To verify the thickness of the samples take z-stacks as the thickness differs equatorially and posteriorly depending on the scleral location. These images show the diverse network of the blood vessel plexis in laminated human sclera derived from the anterior and posterior episclera.

The blood vessels are positive for CD31 as shown in green. In this figure, LYVE1 positive immune cells of the episclera and their relationship to the CD31 positive blood vessels are illustrated. The z-stack series was taken at 10x magnification to examine the three dimensional relationships of blood vessels and immune cells.

As shown here the extra-ocular muscles enclosed within the sclera can also be analysed for the presence of blood vessels or immune cells, using the markers CD31 and LYVE1 respectively. Once mastered the laminating technique can be done in less than one hour if it is performed properly. While attempting this procedure it is important to remember to prevent the tissue from drying out.

Don't forget that working with human tissue can be potentially infectious and precautions such as microbiological and virological diagnostics should always be taken beforehand.