HISTO-RESIN EMBEDDING TISSUE SLICES

from Rick Hyson's protocol. This has been used for tissue slices from 30 to 300 µ thickness.

1. Wash out fixative with buffer, 3 X 30 min.;

2. Dehydrate in 70% ethanol, 2 X 60 min.;

3. Dehydrate in 95% ethanol, 2 X 60 min.;

4. Infiltrate in 1:1 95% EtOH:infiltration solution, 2 hrs.;

5. 100% Histo-Resin infiltration solution, 2 hrs.;

6. Embed.

Embedding:

1. Remove each slice from the infiltrating solution and blot off excess solution;

2. Place a drop of Histo-Resin embedding mixture on a silanized glass slide, one drop

for each slice;

3. Place each slice on a drop of Histo-Resin;

4. Place another drop of Histo-Resin on each slice, and maybe extra resin between slices;

5. Carefully lay another silicon coated slide over the slices so that there are not air

bubbles entrapped;

6. Coat the edges of the "sandwich" with melted paraffin;

7. Allow to cure for 2 hrs.

Note: This method of sandwiching slices between silicon coated glass slides is useful for other methacrylates and epoxy resins, just be sure to use the correct dehydration and infiltration solutions.

The trick with this method is to get enough Histo-Resin between the slides to prevent air bubbles, which will prevent polymerization, but not so much resin that the paraffin cannot adhere to the glass and seal against air.

 d rehydrated;

DO NOT USE POLY-L-LYSINE SUBBED SLIDES FOR EOSIN STAINING. THE PLL PICKS UP EOSIN AND WILL CREATE A PINK TO ORANGE BACKGROUND BEHIND THE TISSUE SECTIONS!

2. Slightly overstain the sections with hematoxylin, usually 3-5 minutes, depending upon section thickness and fixative (up to 20 min. if solution is not fully ripened);

3. Remove excess stain in tap water, 2 min.;

4. Differentiate and destain a few seconds in acidic alcohol until sections look red, usually 4-5 dips;

5. Rinse briefly in tap water to remove the acid;

6. Blue in bicarbonate until nuclei stand out sharply blue, about 2 min.;

7. Rinse in running tap water, 8 min.;

8. Dehydrate and clear, or stain with eosin.

Eosin staining:

SEE NOTE ABOVE REGARDING USE OF PLL SUBBED SLIDES.

1. Take hematoxylin stained slides from the last tap water rinse and place in 70% ethanol for 3 min.;

2. Place slides in eosin for 2 min.;

3. Take slides through 3 changes of 95% ethanol, 5 min. each;

4. Then transfer to the first absolute ethanol of the clearing

series.

 

Testing hematoxylin stain solution to see if it is still usable.

Add several drops of stain to tap water (not distilled or deionized). If they turn bluish-purple immediately, it is satisfactory. However, if they change slowly, stays reddish or brownish, then the stain should be discarded.

tions of the amount and pattern of neuronal