Preparing Acid Washed Glass Cover slips
This helps cells and polyamino acids stick to glass. It also helps your DIC pictures look prettier by getting rid of small particles stuck on the glass.
First separate cover slips from one another and place them into a glass container with a lid containing the amount of water you’ll need to add in order to make a 1M HCl solution. It is important that the cover slips are not sticking to each other because you will need to use one at a time and it’s easier to do this now rather than later.
Be careful when changing solutions not to pour out cover slips into the sink which can both block the drain and be really irritating to some lab members. If you do this, then simply clean them up (be sure to rinse the sink well first if you spill HCl along with the cover slips.
1) Heat cover slips in a loosely covered glass beaker in 1M HCl at 50-60oC for 4-16h.
2) Cool to room temperature
3) Rinse out 1M HCl with ddH2O
4) Fill container with ddH2O and sonicate in water bath for 30 mins
5) Repeat step 4
6) Repeat step 4
7) Fill container with 50% EtOH and 50% ddH2O and sonicate in water bath for 30 min
8) Fill container with 70% EtOH and 30% ddH2O and sonicate in water bath for 30 min
9) Fill container with 95% EtOH and sonicate in water bath for 30 min
10) Fill container with 95% EtOH
Preparation of Squeaky-Clean Coverslips*
1) Place coverslips one at a time in a 500 ml glass beaker filled halfway with hot tap water containing ~5 ml of Versa Clean detergent, taking care to separate coverslips that are stuck together. Sonicate coverslips in hot tap water containing Versa Clean for 45 min in a water bath sonicator.
2) Rinse coverslips 10 times by swirling with hot tap water. Sonicate in hot tap water for 30 min.
3) Rinse coverslips 10 times by swirling with double distilled water. Sonicate in double distilled water for 30 min.
4) Rinse coverslips 3 times by swirling with 1 mM EDTA. Sonicate in 1 mM EDTA for 30 min.
5) Rinse coverslips 3 times by swirling with 70 % ethanol. Sonicate in 70% ethanol for 30 min.
6) Rinse coverslips 3 times by swirling with absolute ethanol. Sonicate in absolute ethanol for 30 min.
7) Rinse coverslips once with absolute ethanol, transfer them to a 500 ml screw cap jar, cover them with absolute ethanol, and store at room temperature until use.
Preparation of poly-lysine coated coverslips
For best results, use high MW poly-L-lysine (greater than 300K).
1. Coat acid-washed coverslips in bulk with 10-15 mL 1 mg/mL PLL in ddH20 in a petri dish. Rock for at least 30 min at room temperature or overnight in the cold room.
2. Wash coverslips in ddH20 at least 10x (free polyaminoacids are cytotoxic!)
3. Rinse coverslips in 100% EtOH and allow to dry by resting on one end in an open tissue culture dish in a sterile incubator or hood. Add cells when dry.
4. For longer storage, allow coverslips to dry on a piece of filter paper. Store dry in a sterile tissue culture dish for several months. Perform step 3 before use.
*=As published in…
Waterman-Storer, C.M. (In press) Microtubule/organelle motility assays. In: Current Protocols in Cell Biology, J.S. Bonifacino, M. Dasso, J.B. Harford, J. Lippincott-Schwartz, and K.M. Yamada, eds. John Wiley, NY.