Placental alkaline phosphatase (PLAP) staining of bladder sections (Version 1.0)

Version

1.0

Notice

This page is the corresponding protocol tomestone page generated as part of the ATLAS-D2K shutdown in July 2025. Many links on this page may be broken.

Authors

Janet Keast

Keywords

[‘histochemical’, ‘cryosections’]

Subjects

[‘Cell biology’]

Release Date

2017-08-07

Abstract

Placental alkaline phosphatase (PLAP) staining of bladder sections protocol by the Keast Group

Procedure

Preparation of tissue for cryosectioning

1. Dissect bladder out fresh from the mouse.

NOTE: Perfusion of mouse can lead to loss of alkaline phosphatase staining

1. Tissue fixation: Embryonic tissue - fix tissue in 4% PFA overnight at 4°C.

Postnatal tissue - fix tissue in 4% PFA for 3-4hrs at 4°C.

1. Wash 3 x 30 mins in 0.1M PBS (pH 7.2) at room temperature.** **

2. Wash and store tissue in 0.1M PBS (pH 7.2) at 4°C (containing 0.1% azide if storing for longer than 1 week).

3. The day before cutting place tissue in 30% sucrose in PBS (0.1M, pH 7.2), leave overnight at 4°C.

4. Embed tissue in OCT and freeze. Cut sections on cryostat, mounting sections on 1% gelatinised slides.

Staining of tissue

1. Air dry slides at room temperature for 30 mins.

2. Wash slides 3 x 10 mins in 0.1M PBS (pH 7.2) at room temperature.

NOTE: For all ages besides adult go straight to step 5. For adult bladder sections perform Steps 3 and 4 to abolish endogenous alkaline phosphatase staining of the urothelium. There may be a small loss of nerve staining in the bladder after treatment with acetic acid.

1. For adult bladder only: Dip slides in 20% acetic acid in dH2O at 4°C for 10 secs.

2. Wash slides 3 x 10 mins in 0.1M PBS (pH 7.2) at room temperature.

3. Heat inactivate endogenous alkaline phosphatase by incubating the slides in pre-heated 0.1M PBS (pH 7.2) at 72°C for 90 mins.

4. Rinse slides in Alkaline Phosphatase Buffer 1 x 5 mins, then 1 x 10 mins at room temperature.

5. Freshly prepare the Staining Solution by adding 20μl of NBT/BCIP Stock Solution (Roche, Cat# 1681451) per 1 ml of Alkaline Phosphatase Buffer. Incubate slides in Staining Solution overnight in the dark at room temperature.

6. Rinse slides 3 x 10 mins in 0.1M PBS (pH 7.2) at room temperature.

7. Coverslip slides in buffered glycerol. Seal edges of coverslip with nail polish.

Solutions

Solutions A and B (below) are used for the making of PBS:

Solution A: (0.2M): 24.0g NaH2PO4 / 1000 ml H2O

Solution B: (0.2M): 28.4g Na2HPO4 / 1000 ml H2O

Phosphate Buffered Saline (PBS) 0.1M (pH 7.2)

1. 140 ml Sol A + 360 ml Sol B + ~450 ml H2O + 8.5g NaCl. Dissolve.

2. pH to 7.2 with 1M NaOH/HCl

3. Make up to 1 litre with H2O

Alkaline Phosphatase Buffer (pH 9.5)

12.11g Tris Base (100mM)

5.84g NaCl (100mM)

10.17g MgCl2.6H2O (50mM)

Make up to 1 litre with H2O

pH to 9.5 with 1M HCl

Consortium

GenitoUrinary Development Molecular Anatomy Project (GUDMAP) Consortium