[Histonet] Preventing Bubbles in Alkaline Phosphatase

[Morken, Timothy]

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?

Images:
http://histosearch.com/imageupload/alkaline-phosphatase-bubbles/

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.


Tim Morken
Pathology Site Manager, Parnassus
Supervisor, Electron Microscopy/Neuromuscular Special Studies
Department of Pathology
UC San Francisco Medical Center
505 Parnassus Ave, Box 1656
Room S570
San Francisco, CA 94143

(415) 353-1266 (ph)
(415) 514-3403 (fax)
tim.morken at ucsfmedctr.org<mailto:tim.morken at ucsfmedctr.org>