Exercise 2.2 - Basophilia

LEVEL I

Materials

• Paraffin sections of Carnoy fixed tissue (liver, kidney)
• Graded series of ethanol, 50, 70, 80, 95 and 100% (v/v)
• Mayer hematoxylin
• Scott solution
• Eosin
• Toluidine blue
• Xylol
• Coverslips and Permount
• Microscope

Procedure

1. Obtain a slide from the instructor. Previously cut paraffin sections have been mounted on slides, and deparaffinized with xylol. They will be brought to the lab in Coplin jars filled with xylol. You will remove the slides from the xylol and transfer them to the indicated solutions which follow:

   Do not let the slides dry at any point in the procedure

2. Transfer the slides sequentially to Coplin jars containing (leave in each solution for the time indicated in parentheses):
   • Absolute Ethanol (1 minute)
   • 95% ethanol (1 minute)
   • 80% ethanol (1 minute)
   • 70% ethanol (1 minute)
   • 50% ethanol (1 minute)
   • Distilled water (1 minute)
   • Mayer hematoxylin (3 minutes)
   • Running water (3 minutes)
   • Scott solution (3 minutes)
   • Eosin (30 seconds)
   • Dip 2X in 95% ethanol
   • Dip 2X in Absolute ethanol
   • Dip 5X in tap water
   • Stain in toluidine blue (10 seconds)
   • Dip rapidly in distilled water
   • Dip once in 95% ethanol.
   • Dip 5X each in two changes of absolute ethanol
   • Place in Absolute ethanol (20 seconds)
   • Place in Xylol (2 minutes)
   • Place in Second Xylol (2 minutes)

3. Remove the slides from xylol, add a drop of permount and a cover slip.

4. Examine the slides with the low (10X) and high dry (40X)objectives of a light microscope. Do not use the oil immersion lens -- the permount will take several days to dry sufficiently and the slides should be treated carefully. If you wish to keep your slides, ask your instructor how to dry them.

   Be particularly careful not to get permount on the lenses.
   

   
   Figure 2.4b Hyperbasophilic region of rat liver.

5. Draw each slide and compare to the commercial preparations of basophilic stained material.

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   Drawing of cells stained with basophilic dyes

This technique was originally designed for the analysis of Mast Cells to demonstrate the differential staining of cells. It will result in cells with pink cytoplasm and blue- -purple nuclei. This is the standard color given to animal cells, just as plant cells are usually dyed green with red nuclei. Of course, the choice of color is purely a matter of personal preference.

With the basophilic stain used here, collagen (an extracellular material) will stain pink, and erythrocytes (red blood cells) will stain red. The procedure is a modification of the standard H&E stain (hematoxylin/eosin) used by most cytologists. The procedures adds the use of toluidine blue, a metachromatic dye. That is, the dye has different colors depending upon the chemical nature of its polymerization. Toluidine blue is a basophilic dye, implying that it stains acid compounds (such as DNA and RNA and acid mucopolysaccharides). Staining with basophilic dyes such as toluidine blue (or methylene blue) is known as basophilia. Intensive staining with these dyes is known as hyperbasophilia and is often used in clinical pathological diagnoses.

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