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Histology Protocols

Dehydration process for embedding tissues into paraffin wax

Tissue should be dissected and placed into tissue prep cassettes appropriate to the size of the sample. Whole frog abdomens and heads require mega-cassettes while liver tissues require smaller cassettes. 1. 70% EtOH I - 60 min 2. 70% EtOH II - 60 min 3. 90% EtOH I - 60 min 4. 90% EtOH II - 60 min 5. 100% EtOH I - 60 min 6. 100% EtOH II - 60 min 7. 100% EtOH III - 60 min 8. Xylene I - 60 min 9. Xylene II - 60 min 10. Paraffin I (58C) - 60 min 11. Paraffin I (58C) - 60 min

Suggestions for embedding tissues into paraffin wax

1. Straight from the dehydrating process, the tissues are placed into wax @ 58C. 2. Samples are placed into embedding molds and arranged into position (desired tissue down). 3. Cassettes are placed onto cold plate (may need to assist the tissue in keeping its position) and an identifying tissue cassette is placed on top. 4. When the wax solidifies the samples can be removed from the mauls and stored at room temperature until further microtoming.

Microtoming Tips
1. Tissues are microtomed at 7 m thick. 2. The number of sections are selected depending on size of tissue (i.e. gonad: one in every ten sections, thyroid: one in every five sections). 3. The sliced paraffin block becomes a fragile ribbon made of wax, as long as preferred (refer to #2), and it is gently placed into warm water bath. 4. While in the water bath the tissues can be separated with the use of a thin dissecting poker, which has been heated with an ethanol lamp. 5. Once the tissue is separated, the section is placed on an albumen slide and placed on a hotplate (you may prefer to check the sectioning quality using a dissecting

microscope to keep a closer eye on the quality prior to staining, look for either dull knife marks or desired tissues). 6. Sections should be placed chronologically in a row down the center of the slide (to facilitate viewing under a microscope)) and left on the hotplate (on low) over night.

Preparing albumen slides

Preparation of Albumin 1. 2. 3. 4. 5. 6. Add two egg whites to 1200mL of DI. Stir on magnetic stirrer for 5 minutes. Add 4mL of concentrated ammonium hydroxide. Stir for 5 minutes. Filter through a low-grade filter (ie: coffee filter). Store albumin in a screw-top glass bottle at 40C in the dark.

Coating Slides 1. Slides are individually laid out on a slide warmer at a low setting (one box of slides can fit on the larger C.S.&E slider warmer). 2. A thin layer of albumin is painted onto each slide using a small clean paintbrush. 3. Slides are allowed to dry on the warming plate overnight at a low setting. 4. Albumin coated slides are stored at room temperature in the original packaging until needed.

Harris' Hematoxylin and Eosin Y Staining Procedure for Paraffin Sections

1. Xylene I - 10 min 2. Xylene II - 10 min 3. 100% EtOH I 10 min 4. 100% EtOH II-10 MIN 5. 90% EtOH - 5 min 6. 70% EtOH I- 5 min 7. H20 - 2 min 8. Hematoxylin - 10 sec 9. Running H20 - 5 min 10. **De-stain if needed: 70% EtOH/HCl solution (until background is clear~7 min) [500 ml 70%EtOH/0.3ml 3M HCl] 70%EtOH II- 2 min 11. Eosin - 1min 12. **De-stain if needed: 95% EtOH (until background is clear~2 min) 13. 100% EtOH III- 2 min 14. 100% EtOH IV- 2 min 15. Xylene III- 2.5min 16. Xylene IV- 3min

17. Permount - add 4 small drops/slide and then carefully place cover slip before the permount dries.

Eosin (pink)- binds to eosinophilic tissue: cytoplasm, muscle fibers(red); connective tissue
fibers; secretory granules; erythrocytes(red); fiberous tissue(pale pink); and protein precipitate.

Hematoxylin (purple) -binds to basophilic tissues: nucleus (proteins and nucleic acids); RNA
and proteins in cytoplasm; mucus and mucoproteins; and cells with large amounts of ribosomal material on smooth ER.