[Histonet] Preventing Bubbles in Alkaline Phosphatase
Timothy.Morken at ucsf.edu
Mon Jul 6 14:39:35 CDT 2015
All knowing Histonet....
We started doing alkaline phosphatase on muscle frozen a little while ago and have had an issue with apparent air bubbles forming over the tissue. Not trapped air from coverslipping, but forming from apparent reaction in the tissue. Does anyone have experience with this and a solution?
We tried longer formalin fixation after the stain (10 min) and an acetic acid rinse after the tap water, after the formalin. Still the same problem
The only mention I have found about this is a Histochemistry text by Lojda from 1979 that says to fix in formalin for several hours after staining to prevent bubble formation. Does anyone have anything shorter? I don't really remember seeing this when I did these stains 20 years ago....
Our procedure (from a method given to our neuropath folks by a group in Australia):
Frozen sections of muscle
Borate Buffer, pH 8.8:
0.992 g Boric Acid
2.28 g Sodium Tetraborate
200 ml distilled water
Mix well. Adjust to pH 8.8. Store at 4°C.
0.1M Magnesium Sulphate
0.6 g Magnesium sulphate, anhydrous (M7506-500G)
50 ml distilled water
Store at 4°C.
ALP Incubation Solution:
15 ml Borate Buffer
2 ml 0.1M Magnesium Sulphate
16.5 mg 1-naphthyl phosphate
16.5 mg Fast Blue RR
Mix in well order. Filter.
Glycerin Jelly Mounting Media
1. Place glycerin jelly in 60°C oven to liquify
2. Air dry slides 15 minutes.
3. Incubate in filtered ALP Incubation Solution at 37°C...45 minutes.
4. Rinse in tap water.
5. Change to 10% formalin...1 min
6. Rinse in tap water.
7. Air dry.
8. Coverslip with Glycerin Jelly.
Pathology Site Manager, Parnassus
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center
505 Parnassus Ave, Box 1656
San Francisco, CA 94143
(415) 353-1266 (ph)
(415) 514-3403 (fax)
tim.morken at ucsfmedctr.org<mailto:tim.morken at ucsfmedctr.org>
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