Lac Z Staining

Lac Z Staining PDF

Modified protocol from Krumlauf Lab.
Also see Methods in Enzymology 225:451-46C-X Tong, B. Chubak

1. Wash the embryos – organs in PBS supplemented with Mg2+&Ca2+ (final conc. 0.5mM and 1mM respectfully) (Dulbecco’s PBS Cellgro; Cat # 21- 030-CV)

500ml PBS-

add 500 µl of 1M CaCl2
add 250µl of 1M MgCl2

2. Fix Embryos – organs: 10-20 min for < 11.5dpc; 30min for>11.5dpc (even 1-2hours)

Glutaraldehyde Fixative

Glutaraldehyde Buffer
5mM EGTA
2mM MgCl2
0.1M NaPi pH 7.3
store @ RT

Add fresh:

A. Glutaraldehyde to 0.1% (v/v) from 50% stock (kept at +4Co) use the fume hood
B. Formaldehyde to 1.5% (v/v) from 37% stock (kept @RT, Laina’s closet)

50ml Fixir:

50µl 50% glutaraldehyde
750µl 37% Formaldehyde

3 Wash 3X30min in PBS/0.02% NP-40 (IGEPAL CA-630; Sigma)

4. Stain for 90 min to ON @ 30oC, or @ 22oC if performing RNA in situ double labeling. Staining solution should be made fresh, can be stored up to 7 days @ +4oC

Solution 12ml total volume
5mM K3Fe(CN)6 0.120ml of 0.5M stock
5mM K4Fe(CN)6 0.120ml of 0.5M stock
2mM MgCl2 0.024ml of 1M stock
0.01% NaDeoxycolate 0.120ml of 1% stock
0.02% NP-40 0.120ml of 2% stock
1 mg/ml X-gal 0.240ml 50mg/ml stock
PBS Up to 12ml

Notes:

-Use 0.04% of NaDeoxycolate for adequate penetration after 14.5dpc

5. Wash 3X with PBS

6. Post-fix in 4% paraformaldehyde in PBS /ON

7. Store/photograph in PBS and 0.1% paraformaldehyde (PFA)

Reagents:

  • X-gal; Roche Cat#651 745. resuspend the powder in N,N-Dimethylformamide (Sigma cat#D-4254) to 50mg/ml
  • EGTA- Ethylene glycol-bis (β aminoethyl ether)-N,N,N’,N’-tetraacetic acid tetrasodium salt, SIGMA E8145
  • K4Fe(CN)6-Potassium hexacyanoferrate (II) trihydrate; SIGMA P9387
  • K3Fe(CN)6- Potassium hexacyanoferrate (III)- Potassium ferricyanide; SIGMA P3667
  • Sodium deoxycolate-Deoxycolic acid C24H39o4Na; SIGMA D6750
  • Glutaraldehyde solution 25% in water, Grade II; SIGMA G6257

Dehydration

  1. Wash the tissues in PBS (∼10 min)
  2. 50% EtOH for 20-30 min
  3. 70% EtOH for 20-30 min
  4. 80% EtOH for 20-30 min
  5. 90% EtOH for 20-30 min
  6. 95% EtOH for 20-30 min
  7. 100% EtOH for 20-30 min, 2X
  8. isopropanol for 20-30 min, 2X
  9. paraffin @ 60oC for 30 min
  10. enbed

Make sections and then contrastain with eosin

Dewaxing

1. isopropanol or histoclear for 5 min, 3X

2. 100% EtOH for 1 min, 2X

3. 95% EtOH for 1 min

4. 70% EtOH for 1 min

Staining

5. eosin 1-5min

Dehydration

6. 70% EtOH for 1 min

7. 95% EtOH for 1 min

8. 100% EtOH for 1 min, 3X

9. isopropanol or histoclear for 1 min, 2X

10. xylene 1’

11. Mounting in Permount (xylene based)