Histology
4 articles
Epoxy Tissue Staining
Biological specimens embedded in epoxy need to be examined by light microscopy, either for orientation prior to further ultra-thin sectioning, or for light microscopy studies. Many histological stains fail to react with the specimen in the epoxy media. Using our E.T.S., sections of tissue embedded epoxy can be stained by a very simple method. This polychromatic staining solution can be used for staining sections of epoxy-embedded specimens prepared for electron microscopy examination, e.g., glutaraldehyde and osmium fixation. The most common application is for sections (0.5 - 1.5µm thick) cut in the ultramicrotome with either glass or diamond knives. The sections are placed on a glass microscope slide and dried on a hot plate. A drop of E.T.S. is added to cover the section on the warmed slide, and the slide is put back on the hot plate until a silver rim is formed. Any excess stain is removed with distilled water from a squeeze bottle, the slide is allowed to dry, and the sections are covered with a cover-slip. The intensity of the stain can be controlled by altering the time that the sections remain on the hot plate. For darker results, allow the stain to dry completely. If sections are over-stained, some of the stain can be removed by washing the section with absolute alcohol. Other methods can be used prior to embedding: En-block, where the specimen is fixed in the usual manner with glutaraldehyde and osmium, rinsed in buffer, and stained for 15 minutes with E.T.S. in a capped vial at 65°C. The specimen is allowed to return to room temperature (10-15 minutes), then dehydrated in ethanol and embedded in epoxy resin. After sectioning of a preliminary block, a secondary staining yields a superior polychromatic colouration for light microscopy. Also, there are no adverse results or artefacts or ultra-structural changes caused by the stain at the E.M. level. The only trace of E.T.S. staining is a slight, bluish colouration of thin sections. A better reaction of this polychromatic stain can be obtained when tissue is fixed with glutaraldehyde alone and not post-fixed with osmium. Reference: Spurlock, B. O. et al, American Journal of Clinical Pathology, Vol. 46 #2, 252 (1966)
Floating Sections
How to stop sections from floating off the grid People have dragged a grid held with old tweezers quickly through a flame. The grid should discolour a little and that often does the trick, causing better adherence of sections to the grid. Very effective is a cellotape solution: Take a meter or two of clear cellotape (20 or 25mm wide), knurl that up and place into a tight jar that is impervious to chloroform. Add ~20ml of chloroform, shake a little. Leave in fridge overnight. Shake again and run through a small filter paper into vial with a tight seal. For use, dip a grid into that solution, touch to the inside of the vial to lose the excess and then place on filter paper. This should be done on the day those grids are going to be used. The used solution will assume a bluish colouring from copper grids - does not matter. The cellotape glue is 'permanently' sticky and will help to keep the sections on the grids. Use chloroform in a fumehood.
Preparing Specimens Without Critical Point Drying
For fast preparation of soft insect tissues, also bacteria and some other "hardy" specimens in lieu of critical point drying Required Hexamethyldisilazane (HDMS) Hepes buffer Method After glutaraldehyde fixation, washing and dehydration through a graded series of ethanol, like: 70%, 85%, 95% and 100%, 5 min in each. Then specimens are immersed in HDMS for 5 min, air dried at room temperature and mounted on stainless steel stubs with double sticky tabs. This treatment gives results as good as those from critical point drying. Its chief advantage lies in the speed with which small pieces of tissue can be prepared for gold coating, compared with the critical point drying technique (about 5 min vs 1.5hr). In addition, the method is inexpensive and does not require special equipment or skills. Slower drying may further reduce shrinkage. Place two filter papers into a glass Petri dish then add a microscope slide. Saturate the paper with HDMS and then place HMDS treated specimens onto the glass slide. Cover with lid and leave to dry overnight. Reference: Nation, J.L. - Stain Technology 58 (6), 347-350 (1983).
Section Handling
When the loop holder (loop could be taped or Araldite to a stick) is held in normal operating position, perhaps 70° from the horizontal, then the loop part should be horizontal. Place the loop under water at the back of the trough, then move to the floating sections and bring the loop up and out of the water. The loop picks up the surface drop of the water, including the sections. Grids for receiving sections should sit (I prefer dull side up) on filter paper. Use a mounted needle or a tine of pointed tweezers to hold the grid down on the rim. Lower the loop onto the grid and the water will be sucked into the paper and the sections positioned nicely on the grid. This method is especially useful if you have problems with folds. Sections may be rounded up on the surface of the trough using a mounted hair. I also like a pointed and mounted Teflon sliver, because sections don't stick to it. The second method is good when you have a single large ribbon to pick up. Orient the ribbon on the water to be in a suitable position. Length of the ribbon can be cut by 'poking' a joint in the ribbon with a Teflon sliver - without the whole ribbon sticking to it, as it may do using a mounted hair. Pick up a grid from a filter paper with good tweezers by the rim, dull side up. While in contact with the filter paper bring the tweezers up into 'handling position' - thereby bending the rim of the grid. Insert the grid at the back of the trough and move then to below the ribbon. Now angle the front of the grid up by about 30° in relation to the horizontal and bring the grid slowly up so that a part of the first section of the ribbon will go down on the under (wrong) side of the grid. As you lift the angled grid vertically from the trough the remaining ribbon will lay across the grid nicely. Touch the grid to a filter paper. Incidentally, a wetted filter paper more easily starts to pull the water from a grid. If the tines of tweezers are wet, you will need to hold the grid down when releasing the tweezers.