TITLE: Preparing fixed cells for immunofluorescence
AUTHORS: Harper JW, Louis R R Hollingsworth, Chan Lee

[DESCRIPTION]
This protocol provides a general method to prepare fixed cells for immunofluorescence imaging, e.g., by confocal microscopy.

[STEPS]
SECTION: Plate coating and cell seeding
1. Dilute 0.1% poly-L-lysine solution (Sigma P8920) in PBS (0.01%). Make enough solution for 0.5 mL per well, plus some excess.
SECTION: Plate coating and cell seeding
2. Add 0.5 mL of the diluted poly-L-lysine solution (0.01% in PBS) to each well of a 24-well glass bottom plate (Cellvis P24-1.5H-N). Place the plate back in the cell culture incubator (37 °C, 5% CO2) for 1 – 24 h to adhere the solution to the plate.
SECTION: Plate coating and cell seeding
3. Wash the wells 3x with sterile PBS or water.
SECTION: Plate coating and cell seeding
4. Add 1 mL of cell culture medium to each well, then plate cells at an appropriate density such that they are not more than 70% confluent on the day of the experiment.
 

SECTION: Fixation and permeabilization
7. Aspirate the cell culture medium and add 1 mL of pre-warmed PBS-PFA to each well. Incubate for 10 mins with gentle rocking at room temperature.
 

SECTION: Fixation and permeabilization
5. Prepare fresh solutions for IF including: PBS, PBST (PBS with 0.02% Tween-20), PBS-Triton (PBS with 0.3%-0.4% Triton-X 100) or 100% methanol pre-chilled at -20 degrees celsius, PBST-Block (see step 5.1 below), and PBS-PFA (see step 5.2 below).
 

SECTION: Fixation and permeabilization
6. Conduct relevant cellular treatment(s). Stagger any treatments so that all of the wells of the plate are ready to harvest simultaneously.
SECTION: Fixation and permeabilization
9. Wash cells 3x with 1 mL PBST. For the first wash, aspirate immediately and replace with PBST, and then conduct the subsequent washes over 5-10 mins.
SECTION: Fixation and permeabilization
8. For Triton-X 100 permeabilization, slowly add 1 mL of PBS-Triton directly to each well then immediately aspirate the solution (this can help prevent the formation of aggregates). Replace with 1 mL PBS-Triton and incubate at RT for 10 min. 

PFA-containing waste should be collected and disposed of properly.
 

SECTION: Fixation and permeabilization
5.2. To prepare PBS-PFA, freshly dilute an ampule of 16% paraformaldehyde (PFA; Electron Microscopy Sciences 15710) in PBS to 4% (add the 10 mL ampule to 30 mL of PBS) in a falcon tube. Warm the solution to 37 degrees celsius prior to fixation.
 

SECTION: Blocking and antibodies
12. The next day, wash wells 3x with 1 mL PBST. For the first wash, aspirate immediately and replace with PBST, and then conduct the subsequent washes over 5-10 mins.
SECTION: Blocking and antibodies
11. Dilute primary antibodies in pre-chilled PBST-Block on ice. Aspirate the wells and add 200 μL of the antibody solution to each well. Rock gently at 4 degrees celsius overnight.

 

SECTION: Blocking and antibodies
10. Aspirate PBST, and add 0.5 mL PBST-Block to each well. Incubate at RT for 1 h.
SECTION: Fixation and permeabilization
5.1. To prepare PBST-Block, dissolve 3% BSA and 22.52 mg/mL glycine in PBST (for example, add 1.5 g BSA and 1.126 g glycine to 50 mL PBST). Glycine binds free aldehyde groups (from PFA) to prevent antibody crosslinking and thus high background, whereas BSA reduces nonspecific antibody binding.
SECTION: Blocking and antibodies
13. Dilute fluorescent secondaries (see materials) 1:200 in PBST-Block.  Phalloidin can also be added at this step. Aspirate the wells and add 200 μL of secondary antibody solution to each well. Rock gently at RT for 60-90 mins.
 

SECTION: Blocking and antibodies
14. Wash cells 3x with 1 mL PBST. For the first wash, aspirate immediately and replace with PBST, and then conduct the subsequent washes over 5-10 mins.
SECTION: Blocking and antibodies
15. Aspirate the PBST and add Hoechst 33342 (0.5 mL, 5 μM in PBS) to each well, if desired. Hoechst labels nuclei in the ~405 channel. Incubate at RT for 10 min.
SECTION: Blocking and antibodies
16. Wash cells 3x with 1 mL PBS. For the first wash, aspirate immediately and replace with PBST, and then conduct the subsequent washes over 5-10 mins.
SECTION: Blocking and antibodies
17. Keep cells at RT if imaging with the same day (ideal), or transfer the plate to 4 degrees celsius for imaging at a later time. Image the plate within ~3 - 5 d with an appropriate confocal microscope.