Sample Fixation

Molecular

Paraformaldehyde Fixation (Best for Gram Negative cells):

Reagents:

4 % Paraformaldehyde:

1. Heat 33ml ddH2O to 60-65°C

2. Add 2g paraformaldehyde while stirring (Use 4g to make a 8% Paraformaldehyde solution)

3. Add 10 N NaOH drop-wise until the paraformaldehyde is completely dissolved.

4. Add 16.5ml of 3xPBS buffer

5. Adjust the pH to 7.2-7.4 at 20°C

6. Filter through a 0.45µm filter

7. Use within 24h or store frozen until ready to use

Protocol:

1. Harvest cells by centrifugation (5600rpm for 5min)

2. Wash the pellet 3x with 1x PBS

3. Resuspend the pellet in 1 part PBS:3 parts Paraformaldehyde solution (i.e.: 250µl 1x PBS + 750µl 4% Paraformaldehyde solution)

4. Incubate overnight at 4°C (Incubate for 3 hours of using an 8% Paraformaldehyde solution)

5. Spin down the cells (5600rpm for 5min) and wash the pellet with 1x PBS (repeat this 3x)

6. Resuspend the cells in 1:1 (1x PBS: 1 ice cold Absolute Ethanol)

7. Store the fixed sample at -20°C.

Ethanol Fixation (Best for Gram Positive Cells):

Protocol:

1. Harvest cells by centrifugation (5600rpm for 5min)

2. Wash the pellet 3x with 1x PBS

3. Resuspend the pellet in 1:1 (1x PBS:1 vol ice-cold Absolute Ethanol)

4. Store at -20°C