Protocol for Staining ENaC Protein

Equipment

Leica CM1950 automatic sectioning cryostat.
Thermo Fisher Scientific round (12 mm diameter) cover glass (thickness No 1.5).
Thermo Fisher Scientific ColorFrostTM Plus slides made of positively charge uncoated glass.

Tissue-tek OCT 4583 compound from VWR.
Tissue-tek cryomolds from VWR.

2% paraformaldehyde in PBS

NaCl 8 g
KCl 0.2 g
Na₂HPO₄ 1.44 g
KH₂PO₄ 0.24 g
Bring to 1 L with dH₂O, and heat to 60-80^oC. Add 20 g paraformaldehyde and stir until it dissolves to give a clear solution. Adjust pH to 7.4. Filter sterilize and store at 4^oC.

30% Sucrose in PBS

NaCl 8 g
KCl 0.2 g
Na₂HPO₄ 1.44 g
KH₂PO₄ 0.24 g
Bring to 1 L with dH₂O, and heat to 60-80^oC. Add 300 g sucrose and stir until it dissolves to give a clear solution. Adjust pH to 7.4. Filter sterilize and store at 4^oC.

NaCl 8.76 g
Tris base 6.05 g
Tween 20 2 ml
Fetal calf serum 50 ml
Bring to 1 L with dH₂O, stir to dissolve and adjust pH to 7.4. Filter sterilize and store at 4^oC.

Mowiol 4-88 2.4 g
Glycerol 6 g
dH₂O 6 ml
Stir for 2hrs at room temperature. Add,
0.2 M Tris 12 ml
Heat to 50^oC for 10 min and stir overnight at room temperature.
Centrifuge the solution to remove precipitates at 5,000 x g for 15min; aliquot and freeze the supernatant at -20^oC.

Freezing tissues

1. Perfuse animal with 2% paraformaldehyde in 1x PBS buffer. Dissect tissues. Cut into small blocks (~5 mm cubic shape) using a scalpel.

2. Dip-fix tissue blocks in 2% paraformaldehyde in 1x PBS buffer at 4^oC for overnight. Transfer tissue blocks to 30% sucrose solution and dip-fix at 4^oC for overnight.

3. Place tissue blocks into Tissue-tek cryomolds and fill the cryomolds with Tissue-tek OCT 4583 compound.

4. Prepare a cooling bath by immersing dry ice in acetone. Float the cryomolds in the cooling bath.

5. Leave for at least 1 min. Remove the cryomolds from the cooling bath with plastic tweezers and transfer directly to a cryostat.

1. Wash ColorFrostTM Plus slides (made of positively charged glass) by dipping in acetone, air dry, and store at room temperature. Store plastic tweezers and brush in the cryostat. Precool cryostat chamber, microtome and a specimen holder (chuck).
(The use of positively charged glass ensures that sections stick firmly to the slides during the immunolabeling and washing steps.)

2. Remove a tissue block from the cryomolds. Place Tissue-tek OCT 4583 compound on precooled cryostat chuck and mount the tissue block onto the chuck so that the side of the cut section is at 90^o to the blade. Cut 6-8 µm tissue sections.

3. Place the slide in a precooled slide box and store in a -20^oC freezer. Cut sections are stable for up to 1 month at -20^oC.

1. Take out the slides from the -20^oC freezer, and dip into a jar filled with ice-cold 2% paraformaldehyde in 1x PBS buffer. Fix at 4^oC for 10 min.

2. Wash slide 3 times with 1x PBS buffer.

3. Make a wet chamber by lining a 24 x 24 cm petri dish with two or three sheets of filter paper and add water to moisten the filter paper.

4. Wash slides once with BLOCK solution. Drain BLOCK solution off against a towel and put slides into the wet chamber.

5. Dilute primary antibody (anti-enac) at 1:100 in BLOCK solution. Add the primary antibody solution to each slide (300-500 µl is sufficient to cover the section on each slide).

6. Incubate the wet chamber at 4^oC for overnight.

7. Take slides out of the wet chamber, drain off antibody, and wash three times with the BLOCK solution.

8. Dilute secondary antibodies (anti-rabbit conjugated to FITC, TRITC or other fluorophores) at 1: 200 in BLOCK solution. Add the secondary antibody solution to each slide and put slides back into the wet chamber.

9.Incubate the wet chamber at 4^oC for overnight.

10. Take slides out of the wet chamber, drain off antibody, and wash three times with the BLOCK solution.

11. Drain BLOCK solution off against a towel. Add 20 ul of Mowiol solution to the section on each slide. Place a cover glass onto the section and apply a small pressure to remove any trapped air bubble.

12. Let Mowiol solution dry at room temperature for 2 hours. Store glass slides at 4^oC for no longer than 1 month.