Single-molecule analysis of replication

Fiber Labeling Protocol (June 2017) 

Pre-Day 1: Split cells into a 6 well plate (or 35 mm dish) so that they will be about 60-70% confluent the next day.  Cells cannot become too confluent or they will not be cycling.  I usually plate 2 x 10⁵ RPE cells in each well.  Plate one extra well so you can harvest non-labeled cells for a dilution later in the protocol.  I like to plate a few extra wells in case of contamination or experimental error the next day.  Equilibrate a flask of media overnight to use for dilutions the next day.  A flask of HBSS should be equilibrated overnight as well.

Day 1 

1. Treat cells with 20 µM CldU for 30 minutes.   
   1. All dilutions should be done using equilibrated media. 
   2. CldU is resuspended in 1 N ammonium hydroxide at 20 mg/mL or 76.1 mM
   3. CldU and IdU are in 20 µL aliquots, stored at -20°C
2. Rinse 2x equilibrated HBSS (2 mLs per rinse). 
3. Treat with damaging agent for desired time. (optional)
4. Wash the wells 2x equilibrated HBSS.
5.  Treat with 100 µM IdU for 30 minutes. 
   1. IdU is resuspended in 1 N ammonium hydroxide at 50 mg/mL or 141.2 mM
6. Wash the wells 2x with equilibrated HBSS. Trypsinize the cells at Room Temperature (300 µL/well for a 6-well plate). 
7. Use ~700 µL media to suspend cells and add to eppendorf tube.  Spin cells down at 2000 rcf for 2-3 minutes. 
8. Aspirate and resuspend cells in ice cold PBS to get about 1x10⁶ cells/mL. 
   1. Dilute labeled cells 1:2 with non-labeled cells before spreading for better results, be sure to mix cells well at a great enough volume.  (stay above 50 µL)
9. Place the slides flat on the apparatus with the white frosted end facing the center. Each apparatus holds 4 slides.
   1. Only label slides in pencil 
   2. Mark slide where cells are added so fibers can be easily found later.
   3. I typically spread 4 different samples on each apparatus since each apparatus tilts the slides differently. Typically, 3-4 slides/sample gives at least a 100 dual-labelled forks. 
10. Add 2 µL of cell suspension to a slide near the white frosted end.  Flick tube to resuspend cells before adding to slide. Leave it to dry ~5-6 min until the sample becomes tacky.
    1. I spread the sample in a vertical line across the slide (instead of placing a drop). This helps “spread” the cells to obtain less overlapping DNA while imaging.   
11. Add 10 µL of spreading buffer to the cells on the slide. Leave for 6 minutes at Room Temperature to lyse the cells. 
    1. Spreading Buffer: 0.5% SDS, 200 mM Tris-HCl pH 7.4, 50 mM EDTA
    2. Add spreading buffer slowly so to keep it from diffusing across slide.
12. Tilt the slide to 15 degrees to allow the DNA to run slowly down the slide.
    1. Make sure drop runs straight and slowly.
    2. Do not allow drop to sit at the top for too long, DNA gets tangled into a ball.
13. Air Dry, at least 40 minutes
14. Fix 2 minutes in 3:1 methanol: acetic acid in a coplin jar. (30ml+10ml)
    1. Do in the hood, very smelly
    2. One coplin jar needs ~40 mLs and holds max of 8 slides
    3. Make fresh and store extra in -20°C
15. Air Dry slides in hood on a diaper.  Takes ~20 mins
16. Store overnight at -20°C for at least a day to a maximum of 5 days
    1. Extra cells in PBS can be kept for less than 6 days

Day 2

1. Treat the slides with 2.5 M HCl for 70 minutes in a coplin jar. {x. 12.1=x. 2.5}
   1. Make fresh, 1 jar needs ~40 mLs
   2. Do in the hood, special waste jar for HCl
   3. Do not treat for less than 25 minutes.
2. Rinse 3x in PBS in the coplin jars
3. Incubate 1 hour in 10% goat serum/ PBSTw (0.1% Triton in PBS) in a coplin jar. 
   1. Sterile filter serum, reduces background staining
   2. Serum can be reused, save and add sodium azide
4. Incubate 2 hours in rat monoclonal anti-BrdU (anti-CldU) {ab6326 9,I8) and mouse anti-BrdU (anti-IdU) {BD347580 8,H4) 1/100 diluted in 10% goat serum/ PBSTw.
   1. Add 100 µL antibody to parafilm and invert slide on top, make sure no air bubbles are present. I typically place the white frosted end first on the drop and leave it gently to allow the drop to diffuse across the slide. 
   2. All incubations are done in the dark, easiest to just stick in a drawer.
   3. Optional: Can use an anti-DNA antibody (mouse) 1/300 to check continuity of DNA fibers. 20 µL aliquots in small -20
   4. Rat monoclonal in 40 µL aliquots in small -20, or box 9
   5. Mouse antibody stored at 4°C in drawer A-H in a small jar
5. Rinse 3x in PBS in coplin jar
6. Incubate 1 hour with secondary antibodies in dark.  Goat anti-rat IgG Alexa Fluor 594 and Goat anti-mouse Alexa Fluor 488 1:350 in 10% goat serum/PBSTw.  100 µL per slide inverted onto parafilm.
   1. 594 antibody: box 8, 4°C, F8
   2. 488 antibody: box 8, 4°C, E1
7. Rinse 3x in PBS
8. Air dry in the dark ~10-15min.
9. Mount with ~110 µL Prolong Gold with no Dapi using whole slide coverslips
   1. Align coverslip with white edge on slide
   2. Minimize air bubbles
10. Let dry overnight in the dark and your slides are ready
    1. Can add nail polish to corners for immediate viewing
    2. After slides have dried overnight at room temperature, place them in a slide box in 4º refrigerator to preserve signal until ready to view.  Recommended to view as soon as possible, or within one week.

• After secondary antibodies are added to the slides, protect the slides from light as much as possible.

Day 3

-Look for fibers under the green or red channel (IdU is green, CldU is red)

- Best to focus on the pencil mark using 20x, then switch to 40x using oil. Fibers will be in the same plane as background specks.  Align objective with the mark made previously on the slide denoting where cells were placed.  Go slow, fibers have a very small field of focus and are easy to pass by. Most of the fibers are detected in the section of the slide towards the frosted end. 

-  About 5 slides per test condition should give enough images.  I usually measure 100 tracks per test condition.

- Fibers that can be measured are straight and have no overlaps with other tracks. 

Reagents

IdU Sigma I7125

CldU Sigma C6891

Rat monoclonal anti-BrdU Abcam ab6326

Mouse anti-BrdU Becton Dickinson 347580

Anti-DNA Millipore MAB3034

Prolong Invitrogen P36930

HBSS: purchased in the molecular cell biology core on 8^th floor of Light Hall

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