Immunofluorescence

Dominik Haudenschild, November 8, 1994 at CBRC

Method I, from Bill Stallcup

For Tissue culture cells on cover-glasses or slides.

1. Wash in 1x PBS

2. Fix in 95% ethanol at -20°C for 10 to 15 minutes. Remove Ethanol, and dry.

3. Block in DMEM or PBS with 2% serum

4. Stain I° antibody diluted in PBS with 0.1% Triton X-100 and 2% Serum for 1 hour at 37°C

5. Rinse in PBS

6. Stain II° antibody diluted as was primary antibody

7. Rinse several times with PBS that contains serum and triton

8. Rinse once with just PBS

9. Cover, and examine under microscope.

Method II, from Matt Schibbler

For Tissue culture cells on cover-glasses or slides.

1. Rinse Medium in 10 gentile dips into PBS

2. Fix cells for 20 minutes with 3% formaldehyde in PBS

3. Rinse off fixative with 25 gentile dips in PBS

4. Permeablize cell in Acetone at -20°C for 7 minutes

5. Stain I° antibody in PBS-Tween with 2% serum at 37°C for one hour

6. Rinse by 25 dips in PBS then 10 minutes in PBS

7. Stain II° antibody at 37°C for 45 minutes diluted as before

8. Rinse by 25 dips in PBS, then 10 minutes in PBS, then 25 dips in PBS, then 50 minutes in PBS. Final Dip is into Milli-Q water.

9. Cover, and examine under microscope.

Method III, from Filippo

1. For Tissue culture cells on cover-glasses or slides.

2. Fix cells in 3% paraformaldehyde in PBS at pH 7.4 with 60mM Sucrose for 30 minutes

3. Wash 3x in PBS

4. Incubate 2 minutes on Ice in PBS with 0.2% Triton X-100

5. Wash 2 times in PBS

6. Wash 1 time in PBS with 0.2% BSA

7. Incubate 1 hour in PBS with 0.2% BSA and I° antibody at 37°C

8. Wash in PBS twice

9. Wash in PBS with 0.2% BSA once

10. Incubate in PBS containing II° antibody and 0.2% BSA at 37°C for 30 minutes

11. Wash ten times in PBS then let sit for 10 minutes in PBS then wash 5 times in PBS

12. Cover, and examine under microscope.

Method IV, from Diana Carlone

For Frozen Sections

1. Section and place on slide

2. Let air-dry for two hours

3. Fix for 30 minutes at room temperature in paraformaldehyde (3.7%) in PBS. TO prepare this heat PBS to 60 degrees C then add the paraformaldehyde. The solution will appear turbid, add a few drops of 10N Sodium Hydroxide until clear. Make fresh every time.

4. Wash 5x with PBS. During the last wash, use PBS with 1% Normal Goat Serum.

5. Observe sections under phase microscope and select best sections.

6. Circle section with rubber cement which is in a 10cc syringe with a needle (19 gauge) with the tip cut off

7. Add primary antibody. IgM use at 1 to 10mg/ml. Incubate for 2 hours at room temperature. However, incubating overnight in a cold, humidified chamber is better.

8. Wash with PBS, three times.

9. Add secondary antibody fro 15 minutes at room temperature

10. Wash 3 times in PBS

11. Mount slides in mounting medium, which is 50% glycerol, 50% PBS. To Prevent loss of fluorescence, you can add N Propyl Galate

12. Add cover slip, and seal with clear fingernail polish

Method V, also from Diana Carlone

Immunofluorescence of paraffin sections

1. Fix tissue in Carnoy's solution overnight at 4°C

2. Transfer tissue to 70% ethanol at 4°C until embedding

3. After embedding and tissues are sectioned, remove paraffin by washing 2x in xylene for 10 minutes each

4. Rehydrate by washing in 100% ethanol for 3x 10 minutes, followed by washes consisting of 95%, 75%, and the 50% ethanol for 10 minutes each.

5. Wash in water for 10 minutes

6. Equilibrate in PBS three times 10 minutes

7. Final wash in PBS with 1% Normal Goat Serum for 10 minutes

8. Incubate in primary antibody diluted in PBS-1% NGS. Use about 100µl of antibody per section which has been circled in rubber-cement, as described above. Incubate in primary overnight at four degrees C.

9. Wash with PBS three times for 10 minutes

10. Incubate in secondary antibody, diluted in PBS with 1% Normal Goat Serum for 45 minutes at room temperature

11. Wash in PBS for three times 10 minutes

12. Remove excess PBS, then add 50% glycerol in PBS.

13. If Fluorescence is good, save slide by sealing all edges of cover-slip with clear fingernail polish.