Materials and Reagents

Mice
Paraformaldehyde (TAAB, catalog number: P001/1 )
Sucrose
Glutaraldehyde (Polysciences, catalog number: 111-30-8 )
Ethanol
Propylene oxide
LR White (London resin, medium)
Hydrogen peroxide
ImmunoSaver (Nisshin EM, catalog number: 333 )
Gelatin
Tween 20
Uranyl acetate
Distilled water or Milli-Q water
Primary antibodies
10-nm gold-conjugated secondary antibodies (we purchased from EY laboratories in Tsuru et al., 2013)
Phosphate buffered saline (PBS) (see Recipes)
0.1 M phosphate buffer (pH 7.2) (see Recipes)
Antigen-retrieval solution (see Recipes)

Equipment

Peristaltic pump
Refrigerator
Freezer
UV irradiator (Nisshin EM, model: TUV-100 ) with 15 W UV light
Gold grid (Maxtaform HR25)
Ultra microtome (Leica Microsystems, model: Ultracut UCT )
Diamond knife (Diatome AG, catalog number: ultra 45 )
Incubator
Microwave (Nisshin EM, catalog number: MWF-2 )
Electron microscope (Hitachi, model: H-7100 )

Procedure

Sample preparation
1. Fix whole mice by perfusion.
  1. Set up perfusion system consisted of needle, tubing, peristaltic pump and beakers with PBS (ice-cold) or 4% paraformaldehyde in PBS (ice-cold).
  2. Anesthetize mice and expose hearts by surgery.
  3. Insert the needle into the left ventricle and make a hole in the right atrium.
  4. Remove blood from mice by perfusion (8 ml/min) with PBS (ice-cold).
  5. Fix mice by perfusion (8 ml/min) with 4% paraformaldehyde in PBS (ice-cold).
2. Isolate colons and cut them up into small pieces (~1.5 mm thick).
3. Fix again with 0.1% glutaraldehyde and 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.2) for 2 h at 4 °C.
4. Wash samples by 0.1 M phosphate buffer (pH 7.2) containing 8% sucrose.
5. Dehydrate samples with a series of ethanol as follows.
  1. 25% ethanol, 10 min at 4 °C
  2. 60% ethanol, 30 minu at -30 °C
  3. 80% ethanol, 30 min at -30 °C
  4. 99% ethanol, 30 min at -30 °C
  5. 99% ethanol, 10 min at RT
  6. 100% ethanol, two changes, 10 min each at RT
6. To promote the infiltration of resin, remove ethanol with propylene oxide as follows.
  1. Ethanol-propylene oxide (2:1), 10 min at RT
  2. Ethanol-propylene oxide (1:1), 10 min at RT
  3. Ethanol-propylene oxide (1:2), 10 min at RT
  4. Propylene oxide, 20 minutes at RT
7. Make resin infiltrate into samples by immersion as follows.
  1. Propylene oxide-LR White containing accelerator (3:1), 2 h at RT
  2. Propylene oxide-LR White containing accelerator (1:1), 2 h at RT
  3. Propylene oxide-LR White containing accelerator (1:3), 2 h at RT
  4. LR White containing accelerator, 12 h at RT
8. Polymerize resin under UV irradiation for two days at RT.
9. Cut the embedded samples (90 nm thick) with ultra microtome and mount on uncoated gold grids (300 mesh).
Immunostaining
1. Treat sections with 3% (vol/vol) hydrogen peroxide for 30 min at RT to unmask epitope.
2. Wash sections with distilled water.
3. Treat sections with antigen-retrieval solution for 15 min under microwave at 95 °C by interval of 2 sec.
4. Block sections with 0.1% gelatin in PBST (PBS containing 0.05% Tween 20) for 30 min at 37 °C.
5. Reacted sections with primary antibodies diluted with PBST overnight at RT.
6. Wash sections three times with PBST for 10 min each at RT.
7. React sections with 10-nm gold-conjugated secondary antibodies diluted with PBST for 2 h at RT.
8. Wash sections twice with PBST for 10 min each at RT.
9. Wash sections twice with distilled water for 5 min each at RT.
10. Stain sections with 4% uranyl acetate for 10 min at RT.
11. Wash sections with distilled water.
12. Observe sections with an electron microscope.

Recipes

Phosphate buffered saline (PBS)
Dissolve 2.89 g of Na₂HPO₄·12H₂O, 0.2 g of KH₂PO₄, 8.0 g of NaCl, and 0.2 g of KCl in distilled water
Adjust the final volume to 1,000 ml
Sterilize by autoclaving
0.1 M phosphate buffer (pH 7.2)
Solution A: Dissolve 13.61 g KH₂PO₄ in 1,000 ml distilled water
Solution B: Dissolve 17.8 g Na₂HPO₄·2H₂O in 1,000 ml distilled water
Mix appropriate volumes of solution A and solution B to adjust pH to 7.2
Antigen-retrieval solution
Dilute ImmunoSaver 200-fold with distilled water

Acknowledgments

This work was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grants 24228002, 24248019 (to K.K.), and 14580699 (to A.T.), Ministry of Education, Culture, Sports, Science and Technology in Japan (MEXT) KAKENHI Grant 19058010 (to K.K.), The Uehara Memorial Foundation (K.K.), Takeda Science Foundation (K.K.), Mitsubishi Foundation (K.K.).

References

Tsuru, A., Fujimoto, N., Takahashi, S., Saito, M., Nakamura, D., Iwano, M., Iwawaki, T., Kadokura, H., Ron, D. and Kohno, K. (2013). Negative feedback by IRE1β optimizes mucin production in goblet cells. Proc Natl Acad Sci U S A 110(8): 2864-2869.