Acid Washing Coverslips

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WARNING: These procedures involve the use of heated strong acids. Clearly label all containers with warning signs, and wear all appropriate safety equipment.

Acid cleaning coverslips is critical for microscopy-based experiments. Cleaning coverslips can improve cell adhesion, and removes potentially autoflourescent materials from the cover slips. Acid cleaning is strongly encouraged for conventional immunoflourescence and is required for single particle tracking and super-resolution microscopy.

Hydrochloric Acid Washing

This is the simplest method for cleaning coverslips and is sufficient for most applications. This procedure should be carried out in a lidded glass beaker.

  1. Rinse coverslips 3x with distilled water
  2. Soak coverslips for 15 min in 70% ethanol with frequent agitation
  3. Rinse coverslips 3x with distilled water
  4. Cover coverslips with 2M HCl diluted in distilled water, cap beaker and place on a hot plate set at 70C - the acid should reach a temperature of 60-65C.
  5. Heat for 2 hours with frequent agitation. Coverslips can be left in acid over night if required.
  6. Carefully transfer beaker to the sink and flush with 50 volumes of distilled water
  7. Rinse with 70% ethanol
  8. In a running tissue culture hood, use tweezers to place coverslips on kimwipes to dry.
  9. Once dried, use tweezers to transfer coverslips to an autoclaving container. It is recommended that pieces of kimwpies be placed between coverslips to prevent sticking.
  10. Autoclave using dry cycle.

Nitric Acid Washing

This is a more robust washing method that removes more contaminants than hydrochloric acid washing, but is also much more dangerous. This procedure should be limited to cleaning coverslips for experiments where no residual organic material can be tolerated.

  1. Rinse coverslips in water and ethanol, as per steps 1-3 in the hydrochloric acid method, above
  2. Soak coverslips in 6M to 10M nitric acid for 20 min
  3. Using tweezers, remove the coverslips from the acid bath and wash 8 times with double-distilled water
  4. Dry coverslips in a tissue culture hood and steralize as per steps 8-10 in the hydrochloric acid method, above.
  5. Neutralise and dispose of the acid using appropriate methods.
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