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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.
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