半乳糖苷酶和碱性磷酸酶染色方法Histochemical Staining

This protocol has been optimized for b-galactosidase and human placental alkaline phosphatase staining in retinal tissue and cultured cells.

Solutions

0.5% Gluteraldehyde/PBS

25 ml 1X PBS

0.5 ml 25% gluteraldehyde (Sigma)

20% Paraformaldehyde/4% Paraformaldehyde-PBS

200 g paraformaldehyde

1 ml 10N NaOH

up to 1 liter with Q, heat to 65° to dissolve, aliquote and store at -20°

Mix 100 ml 20% paraformaldehyde with 50 ml 10X PBS and bring up to 500 ml with Q

filter, and store at 4° for up to 2 weeks

10X PBS

80 g NaCl

2 g KCl

11.5 g Na2HPO4

2 g KH2PO4

up to 1 liter with Q

30% Sucrose/PBS

150 g sucrose

50 ml 10X PBS

up to 500 ml with Q

sterile filter and store at room temperature

AP Detection Buffer

100 mM Tris 9.5 5 ml 2M Tris 9.5

50 mM MgCl2 5 ml 1M MgCl2

100 mM NaCl 2 ml 5M NaCl

up to 100 ml with Q

store at room temperature

100X NBT

0.75 g NBT (Sigma #N6639)

up to 10 ml with 70% DMF

store at -20° in a light proof bottle

100X BCIP

0.5 g BCIP (Sigma #B6777)

up to 10 ml with DMF

store at -20° in a light proof bottle

100X X-gal

1 g X-gal

10 ml DMF

store at -20° in a light proof bottle

b-galactosidase Rinse A

100 mM NaPO4 pH 7.3 mix monobasic and dibasic

2 mM MgCl2 2 ml 1M MgCl2

5 mM EGTA 10 ml 0.5M EGTA pH 8.0

up to 1 liter with Q

store at 4°C

b-galactosidase Rinse B

100 mM NaPO4 pH 7.3 mix monobasic and dibasic

2 mM MgCl2 2 ml 1M MgCl2

0.01% Na deoxycholate 1 ml 10% Na deoxycholate

0.02% NP-40 1 ml 20% NP-40

up to 1 liter with Q

store at 4°C

b-galactosidase Developer

100 mM NaPO4 pH 7.3 mix monobasic and dibasic

2 mM MgCl2 2 ml 1M MgCl2

0.01% Na deoxycholate 1 ml 10% Na deoxycholate

0.02% NP-40 1 ml 20% NP-40

5 mM K3Fe(CN)6 1.6 g K3Fe(CN)6

5 mM K4Fe(CN)6 2.1 g K4Fe(CN)6

up to 1 liter with Q

store at 4°C

Procedure

beta-galactosidase staining

• Fix the tissue on ice for 5-10 minutes in 0.5% Gluteraldehyde/PBS.

• Wash 3 times in PBS, and rinse briefly in Rinse A. Incubate in Rinse A for 30 minutes.

• Rinse briefly in Rinse B and incubated in Rinse B for 5 minutes.

• Incubate in Developing Solution containing X-gal for 30 minutes-4 hours at 37°.

• Stop the reaction by rinsing for 5 minutes in PBS and repeat several times.

alkaline phosphatase staining

• Fix the tissue in 4% paraforaldehyde/PBS for 5 minutes on ice and rinse twice in PBS.

• Heat to 65° for 30-60 minutes.

• Cool, and rinse once in AP detection buffer to raise the pH.

• Develop in AP detection buffer containing NBT and BCIP overnight at room temperature in a light proof container.

• Stop the reaction by washing 3 times in PBS pH 5.5.

NOTE: if both b-gal and AP detection are going to be performed on the same tissue then perform b-gal first.