Histology - Wax post OPT
Embryos set in agarose for OPT had the agarose removed, were cleared and set in wax to be serially sectioned.
Protocol
Previous preparation
Embryos were previously prepared in agarose.
Embryos embedded in agarose were OPT scanned.
Removal of agarose
Cut the agarose block off magnetic block. Trim/cut agarose to make as small as possible and wash in methanol - at least 3 o/n methanol washes to remove residual BABB.
Move the agarose block through ethanol gradient 70% - 10% to 0.29M sucrose leave several hours to equilibriate and then leave in 0.29M sucrose overnight at 4oC.
Place the agarose block at 58 - 60oC for 1hour in prewarmed 0.29M sucrose. The agarose should melt in 10 - 15 minutes.
The remove the embryo with care.
Rinse 0.29M sucrose
Up ethanol gradient 10% 30% 50% 70% 90% (Can store embryos in 70% at this point)
Dehydration, infiltration and embedding.
Post removal of agarose, specimens were taken from 70% ethanol up to 100% ethanol in a graded series. They were cleared in xylene then infiltrated and embedded in paraffin wax (56 degree melting point) in moulds suitable for the specimen size and orientation noted.
Sectioning directly onto water.
The selected wax block was trimmed, mounted on a chuck and attached to the microtome (Leica RM2264 motorised with retraction and microscope attachment). Serial sections were cut at 7um, using the motorised setting, directly onto distilled water in a trough specially designed and manufactured 'in-house' to fit the disposable knife holder. This method of sectioning eliminates static, is cleaner and results in greater consistency of flattening of the sections as they leave the knife edge. Section loss is also kept to a minimum.
| microtome sectioning |
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The strings of sections were removed from the trough by sliding a clean wet glass microscope slide under the surface of the water below the sections. The slide with the strings of sections are gently introduced into a floating-out bath at 42-45oC. The sections were floated off the slide onto the surface of the water where they were allowed to stretch out for a few seconds (20-30secs.)
The time and temperature were carefully monitored to give consistency to the stretching. The strings were then aligned on the slide as it was withdrawn from the water and any repositioning carried out using a needle before the excess water was drained away. The slides were then placed vertically on a rack and allowed to dry at room temperature overnight.
Staining
Before staining, slides were placed in an oven at 60oC for 2 hours. Slides were then de-waxed in xylene and rehydrated through a graded series of ethanol/water mixes.
After washing in running water, slides were stained in Mayer's Haematoxylin 5 mins. and freshly prepared Eosin 3mins.,dehydrated,cleared in xylene and mounted in DPX.
| Prepared slides |
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Image capture
After staining, the slides were available for image capture.
