Exercise 2.6 - Alkaline Phosphatase Localization

LEVEL II

Materials

• Kidney sections (preferrably frozen sections)
• Cold acetone
• Chloroform
• Incubating Solution

  0.8% (w/v) paranitrophenyl phosphate	2.0 ml
  2% (w/v) sodium barbitol	2.0 ml
  distilled water	1.0 ml
  2% (w/v) calcium chloride	4.0 ml
  5% (w/v) magnesium sulfate	0.2 ml

• Incubator at 37° C
• 2% (w/v) cobalt nitrate
• 2% (w/v) ammonium sulfide
• Acetone solutions 100, 80 and 50% (v/v) in water
• Mayer's hematoxylin
• Scott solution
• Ethanol series, 50, 70, 95 and 100% (v/v)
• Xylol, Permount, slides, coverslips
• Microscope

Procedure

1. Use a cryostat to cut 10-15m frozen sections of kidney at -2° C. Mount on gelatin coated slides and dip in cold acetone to fix. Alternatively cut 10m paraffin sections of acetone or formaldehyde fixed materials. Acetone fixation will result in better enzyme preservation than formaldehyde. Tissues should be fixed in chilled acetone for 24 hours prior to paraffin embedding. Low temperature paraffin (560 melting temperature) works best and 10m sections need to be deparaffinized with chloroform before hydration through a series of acetones to water.

2. Incubate in incubating solution, 37° C for 30 minutes.

3. Wash in distilled water.

4. Place the slides in 2% cobalt nitrate for 5 minutes.

5. Rinse in distilled water.

6. Place the slides in 2% (w/v) yellow ammonium sulfide for 2 minutes. This step should be done in a ventilated hood to avoid the sulfide fumes.

7. Wash in distilled water.

   The slides can be viewed at this point with phase contrast or as a simple wet mount. If there is not sufficient black sulfide precipitate, the slides can be returned to the ammonium sulfide for an additional 2 minutes. If further contrast is needed, the cells can be counterstained with any basophilic stain. The following uses Mayer

   • Place in Mayer's hematoxylin (3 minutes)
   • Place in running water (3 minutes)
   • Place in Scott solution (3 minutes)

8. Dehydrate by passing through 50, 70, 95 and 100% ethanols, clear with xylol and mount a coverslip with permount.

9. Examine all slides and compare the distribution of alkaline phosphatase on the surface of the cells.

   Sites of alkaline phosphatase will be black, sharp and clear, nondiffuse. The reaction precipitates calcium phosphate as the phosphate is released by the enzyme. The calcium phosphate in turn is turned to black (or brown) prepicipate in the presence of the cobalt nitrate and ammonium sulfide.

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