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Sample Preparation for Confocal Microscopy 

 


Note: These are generic protocols. Every different antibody and each different tissue type will require fine tuning many of these steps (at least!).


Index: Click on a heading to jump directly to that section.

Complete Generic Cell Preparation Protocol

Complete Generic Tissue Preparation Protocol

            1. Paraformaldehyde Fixation             DAB -Peroxide staining
            2. Staining
            3. Mounting

                            

               

                            

 


 

Part 1: Cell monolayers

Paraformaldehyde Fixation

  1. Prepare a fresh 4% solution of paraformaldehyde by heating in buffer (PBS, TRIS, HEPES, Veronal, etc.) to 55- 60 C. Add several drops of 1N NaOH and stir until dissolved.  Allow cooling to room temperature before use.

2.     Wash the coverslips once with PBS and then fix with PFA for 15min.

3.     Wash the coverslips once with PBS and then permeabilise the cells with 0.2% Triton-X100 in PBS for 5min. 

4.      Wash the cells once with PBS and then quench in fresh 0.1% sodium borohydride in PBS for 5min. 

5.      Wash three times in PBS and proceed to staining protocol.

     

Staining

 1.      Dilute primary antibody as appropriate in 1% BSA in PBS.  Centrifuge the diluted antibody for 5 min at 12,000 x g in a refrigerated microcentrifuge prior to use will remove aggregated material and reduce background.

 2.      Incubate the coverslip with a small volume of diluted primary antibody for 1h.

 3.      Dilute the secondary fluorescent antibody as appropriate in either PBS or 1% BSA in PBS, depending on the background staining.  Again, centrifuging the diluted antibody for 20 min at 12,000 x g in a refrigerated microfuge will reduce background.

 4.      Incubate the coverslip with a small volume of diluted secondary antibody for 45min.

 5.      Wash coverslips three times with PBS.

 

Mounting

Mount in appropriate mounting media containing an anti-photobleaching reagent (glycerol, Mowiol, BABB etc. with 0.6% DABCO, p-phenylenediamine, etc). Coverslip, seal with nail polish and store in the dark at 4 degrees C.


 

 

 

 

Part 2: Tissue Samples

Note: This procdure was worked out using MF20 (mysosin heavy-chain specific) and M1B4 (tenascin specific) on chick embryos.  This protocol may need adjustment for use with other ab's.

 1.      Fix embryos in 4% PF o/n at 4 C.

2.      Rinse 2X with PBT or PBS, 5 each.

3.      Dehydrate  with a MeOH series 5 each step.

4.      Bleach o/n at RT in Dents bleach (1 part DMSO; 4 parts MeOH with 10% hydrogen peroxide). This step also inactivate endogenous peroxidase activity.

5.      Wash 3X in PBS or PBT, 1h each.

6.      Incubate with primary ab in 5% serum, 20% DMSO, in PBS o/n at RT.

7.      Wash 5X with PBS, 1h each.

8.      Incubate with secondary ab in 5% serum, 20% DMSO, in PBS o/n at RT.

9.      Wash 5X with PBS, 1h each. (Can stop here and store at 4 C o/n).

10.  For DAB detection, do the following:

11.  Make Vectastain A/B complex while doing the last wash of step 9. (Use the regular ABC kit: add 2 drops of A solution and 2 drops of B solution in 10 mL PBT; let solution sit for 30 before use). 1 drop=50mL

12.  Incubate with A/B mix for 30.

13.  Wash 3X with PBS, 1h each.

14.  React with DAB/hydrogen peroxide.


 

 

 

 

DAB/Peroxide

1.   Dilute 10 mg/ml DAB (Stock) to 1 mg/ml in 0.1 M Tris pH7.2; that is add 0.5 mL of DAB stock (kept at -20 C) in 4.5 mL of Tris buffer.

2.   Dilute an equal volume of 30% hydrogen peroxide to 0.03% in distilled water (that is 5 mL in 5 mL of water).

3.   Mix the two solutions together and use immediately. Use 2-3 mL per vial.

 -         Detection will take 5-10 at RT.         

4.   Stop reaction with PBS. Wash 3X with PBS or PBT 10 each.

10. If using fluorescence probes, follow MeOH series after step 9.

11. Clear in BABB (benzyl alcohol / benzyl benzoate 1:2).

12.  Store containers in dark.


 

 

 

 

Paraformaldehyde - Triton  X-100 Fixation

1.  Briefly rinse cells with PBS at 37C.

2.  Fix samples with 4 % paraformaldehyde in PBS for 10 minutes at RT.

3.  Wash samples 3x with PBS, for two minutes each time.

4.  Permeabilize cells with 0.2% Triton X-100 diluted with PBS, for 5 minutes.

5.  Rinse samples 4x with PBS, for two minutes each.


 

 

 

 

 

Paraformaldehyde - Acetone Fixation

1.  Briefly rinse cells with PBS at 37C.

2.  Fix cells with 4 % paraformaldehyde in PBS for 10 minutes at RT.

3.  Wash cells 3x with PBS, for two minutes each time.

4.  Permeabilize cells with -20C acetone for 10 minutes (do in -20C freezer).

5.  Either:       a)  airdry cells and store, or

b) Rehydrate cells in PBS for 10 minutes at room temperature.


 

 

 

 

 

Methanol - Glacial Acetic Acid Fixation

1.  Add one volume 3:1 methanol - glacial acetic acid to one volume of the growth medium covering the cells. Incubate for five minutes at room temperature.

2.  Wash cells briefly in fresh 3:1 methanol - glacial acetic acid, and fix for an additional 10 minutes in fresh 3:1 methanol - glacial acetic acid.

3.  Rinse 2x with distilled water for 5 min.


 

 

 

 

 

 

Acetone fixation

1.  Briefly rinse cells with PBS at 37C.

4.  Fix cells with -20C acetone for 10 minutes (do in -20C freezer).

3.  Either:      a)  airdry cells and store, or

b) Rehydrate cells in PBS for 10 minutes at room temperature.


 

 

 

 

 

 

Acetone - Triton X-100 Fixation (via Molecular Probes, Inc.)

1.  Rinse cells 2 times with HBSS at 37C.

2.  Fix cells by incubation in acetone at room temperature for five minutes.

3.  Rehydrate cells for 10 minutes in PBS.

4.  Permeabilize cells further by incubation in 0.2% Triton X-100 in PBS for five minutes.

5.  Rinse samples 34 times, for one minute each time, in PBS.


 

 

 

 

 

 

 

Methanol - Acetone Fixation

1.  Briefly rinse cells with PBS at 37C.

4.  Fix cells with a 50-50 mixture (v/v) of -20C acetone - methanol for 10 minutes (do in -20C freezer).

3.  Either:       a)  airdry cells and store, or

b) Rehydrate cells in PBS for 10 minutes at room temperature.

 


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