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Mouse Embryonic Tissue

Protocol for Nancy Speck's lab, Biochemistry Dept.

References
Marin-Padilla, M. and M. Amievo (1989) "Early neurogenesis of the mouse olfactory nerve: golgi and electron microscope studies" J. of Com. Neurology 288:339-352

Sekerkova, G. et al 1997, "Visualization of b-Galactosidase by enzyme and immunohistochemistry in the olfactory bulb of transgenic mice carrying the LacZ transgene", J. of Histo. And Cytochemistry 45(8) 1147-1155

PRIMARY FIXATION after enzyme stain: 3% GTA, 2% PF, with 2.5% DMSO and a 0.1% CaCl2 buffered by 0.1 M Na Cacodylate Ph 7.4.

Sucrose ------ MW -------- 342.3 gms

Na Cacodylate(.3H20) - MW ------------- 214 gms

Stock Buffer: 0.2 M Na Cacodylate containing 0.1% CaCl2 and 0.6 M Sucrose at Ph 7.4 :

4.28 NaCac., 0.1 gms CaCl2, 20.4 gms Sucrose(MW=342.3) in 100ml dH20

1a. Pregnant mouse was sacrificed:

2. Embryonic aortic tissue area was pre-fixed, cut and stained with Bluo-gal or X- Gal(Sekerkova et al (1997). In Summary: whole embryo is placed in pre-fix for 10-15 mins(0.25%GTA/2%PF in 0.1M Pipes pH7.4); then head is removed and rest of embryo is place in fresh pre- fix for 1.5 hrs. total fix time at RT.

Bluo-gal or X-Gal stain until blue appears in aortic area (2-6 hrs.)

3. Tissue was postifixed in 3% GTA, 2% PF, with 2.5% DMSO and a 0.1% CaCl2 buffered by 0.1 M Na Cacodylate Ph 7.4. further fixed for at least three hours at room temperature Sometime during fixation, another cut is made to select area of aortic tissue for viewing(fresh fixative is added at this time). Embryos can then be stored overnight at 4 C or for 36 hours until DNA tests are complete before further processing.

4. Tissue blocks were rinsed in 0.1 M Na Cacodylate at Ph 7.4; 3X; 15 min. each.

5. SECONDARY FIXATION: 2% OsO4 in 0.1 M Na Cacodylate at Ph 7.4.

Tissue blocks were immersed in secondary fixative for 2 hours at RT.

1b. Pregnant mouse was sacrificed: Ultrastructural study:

2. Embryonic aortic tissue area was fixed in3% GTA, 2% PF, with 2.5% DMSO and a 0.1% CaCl2 buffered by 0.1 M Na Cacodylate Ph 7.4. In Summary: whole embryo is placed in Fix for 10-15 mins; then head is removed and rest of embryo is transfered to fresh fix and further fixed for at least three hours at room temperature. Sometime during fixation, another cut is made to select area of aortic tissue for viewing(fresh fixative is added at this time). Embryos can then be stored overnight at 4 C or for 36 hours until DNA tests are complete before further processing.

3. Tissue blocks were rinsed three times in 0.1 M Na Cacodylate containing 0.05% CaCl2 and 0.3 M Sucrose at Ph 7.4.

4. SECONDARY FIXATION: 2% OsO4 in 0.1 M Na Cacodylate containing 0.05% CaCl2 and 0.3 M Sucrose at Ph 7.4.

5. Tissue blocks were immersed in secondary fixative for 2 hours at RT.

6. Tissue blocks were rinsed in 0.1 M Na Cacodylate at Ph 7.4.

7. Rinse in dH2O.

8. Dehydrate thru ETOH series: 30%, 50%, 70%, 95%, 15 mins. ea.

9. Three 15 min. changes in 100% ETOH.

10. Two 10 min. changed in Propylene oxide(PO).

11. Bluo-gal/X-gal tissue blocks were immersed in Epon-Araldite 1:1. Cap vials for 2 hrs. Remove caps and place in vacuum desiccator overnight

Ultrastructural tissue blocks were immersed in LX112:PO - 1.5:1 {10.2 ml:6.8 ml}. Cap vials for 2 hrs. Remove caps and place in vacuum desiccator overnight

12. Transfer tissue blocks to flat molds. Orient and fill Bluo-gal/X-gal tissue blocks with fresh Epon-Araldite. Orient and fill Ultrastructural tissue blocks with fresh LX112. Place in vacuum desiccator overnight.

13. Polymerize:

Ultrastructural tissue blocks 60 C for 48 hrs.

Bluo-gal/X-gal tissue blocks 60 C for 24 hrs

Remove blocks from heat to cool.

EPON/ARALDITE:

10ml. LX112(POLYBED812, etc.)

10ml. Araldite 502

24ml. DDSA

0.9ml DMP 30 (added just before use)

Mix together first three by shaking vigorously. Warm in 50?C oven for 5-10 minutes, to improve mixing. Add DMP-30 and mix vigorously. Resin mix may turn orange, depending on pH of DMP-30. Remove air bubbles with vacuum for @ 30 minutes. Store aliquots of resin mix in 5 or 10ml disposable syringes in -20?C freezer; will keep for 1 week. Be sure to use same resin mix for all steps of infiltration.

SECTIONING:

1. A. Cut 0.5 ? sections and stain LX112 sections with "Epoxy Tissue stain"(Toluidine blue and Basic Fuschin from EMS Co.). stain section, heat, rinse and dry.

This stains a light pink color and allows for blue enzyme stain to show through.

B. Cut 0.5 ? sections and stain Epon/Araldite sections with 1% Acid Fuschin in dH2O. Stain section at @180oC on hot plate for 1 minute. This stains a light pink color and allows for blue enzyme stain to show through.

C. Cut 0.5 ? sections and stain LIGHTLY with Methylene Blue/Azure II. This is for Ultrastructure.

A. 1% Methylene Blue in 1% Na Borate (Borax)

B. 1% Azure II in H2O

Mix fresh A + B 1:1 - stain section, heat, rinse and dry.

2. Stain thin sections: Must use UAaq to avoid creating holes in Bluo-gal/X-gal tissue by methanolic UA, which can dissolve stain!

Bluo-gal/X-gal/Epon-Araldite tissue sections: 2%UAaq for 9' & Reynold's LC for 2'

or Reynold's LC for 3' with no Ua stain

Bluo-gal/X-gal/LX112 tissue sections: 2%UAaq for 50' & Reynold's LC for 8'

or Reynold's LC for 8' with no Ua stain

(this is for previous Bluo-gal blocks #188, #215,#220,#227)

Ultrastructuralfix/LX112 tissue sections:2%UAmeoh for 20' and Reynold's LC for 3-5'.

Last Updated: 10/2/08