Materials 37% formaldehyde: for
10 mL, add 3.7g p-formaldehyde to 5mL PEM. Mix and heat to 70 degrees
(or when you can start to see condensation on the side of beaker). Add 10N NaOH dropwise
(about 1.5mL total)until almost all of the formaldehyde has dissolbed but the solution is still little
cloudy. Make up to 10mL with PEM.
PEM: for 500mL, 100mM PIPES , pH 6.9 (50mL); 1mM EGTA (1mL); 1mM MgSO4 (0.5mL)
PEMS: for 200mL, PEM; 1M sorbitol (36.43g)
PEMBAL: for 200mL, PEM; 0.1M L-lysine (3.65g); 1% BSA (2g); 0.1% sodium azide (0.2g)
1. Grow up culture of interest in 50mL YES (in autoclaved
250mL flask) and shake overnight. Following morning,
check by microscope to see that cells are dividing (optional). Measure the OD(595). The ideal OD is 0.8.
2. Make fresh 37% formaldehyde. Fix cells by adding
1/10th volume (5mL) of 37% formaldehyde to the culture. Put
flask back and shake at culture temperature for 60-90 min.
3. Spin cells at 2k for 5 min. Transfer to eppendorf tubes
with 1mL PEM and wash 2x more. (Note: when spinning
down, do it for 15s at 5k twice--once upright, second time rotating it 180 degrees.)
4. Resuspend in 1mL PEMS with 0.5mg Novozym (10uL stock)
and 0.5mg Zymolyase (10uL stock). Incubate at
30 degrees until >90% cells are digested (usually 25-30 min.). Check by adding 1uL 10% SDS to 9uL cells and check
under microscope for absence of cell wall).
5. Wash 3x with 1mL PEMS. Resuspend in 1mL of 1% Triton/PEM for 2 min.
6. Wash 1x with 1mL PEM.
7. Roughly assess the volume of final pellet. Resuspend
in 1mL PEMBAL and transfer a volume which will give
20-30uL pellet to each eppendorf tubes, filling up to 250uL with PEMBAL if necessary. (Usually OD of 0.8 corresponds
to dividing once to two 500uL and OD of 0.4 usually requires no transfer--however, assessment by eye is the most
important indicator.) Put on rotating wheel at RT for at least 30 min.
8. Spin down. Resuspend in 150uL PEMBAL with 1:100 primary Ab (1.5uL).
9. Incubate 16h (overnight) at RT on rotating wheel. END OF DAY ONE.
10. Wash 3x with 1mL PEMBAL. Resuspend in 1mL PEMBAL and rotate on wheel for at least 30 min.
11. Resuspend in 150uL PEMBAL with 1:100 secondary Ab
(1.5uL)-->use aluminum foil to cover tubes as they are light
sensitive from this point on.
12. Repeat step 9. END OF DAY TWO.
13. Repeat step 10.
14. Resuspend pellet in 150 uL PEMBAL and mount on poly
L-lysine coated coverslips (which is done by covering coverslips
with poly L-lysine solution for about 5 min. and washing it away). Taking 50uL cells, cover the coverslips and taking away the
excess. Let it sit for 5 min. (from now on, should be under the bucket as they are light sensitive) and wash it away. Cover with
DAPI (1:100,000 dilution of 1mg/mL stock) for 5 min. and then wash with water for 5 min. Put 5 uL glycerol on microslides (which
should be washed with 70% ethanol) and invert dried coverslips onto them. Seal with nail polish and view under microscope.