Immunofluorescence with J2 antibody

J2 antibody immunofluorescence

Cell Plating and Knockdown:

1. Place 12mm circular cover slips into a 24-well plate
2. UV sterilize for 10min
3. Wash once with DMEM media
4. Harvest HeLa cells and count
5. Dilute cells to 40K/ml and plate 1ml of cells per well
6. During trypsinization, mix:
   1. 50ul of OptiMEM with 1ul of RNAimax per sample (Tube A)
   2. Incubate at RT for 5 min
   3. 50ul of OptiMEM with 1ul of Control or SETX siRNA per respective samples (Tube B)
   4. After 5min, add Tube A to Tube B, mix and incubate at RT for 20 min
      1. Split Tube A in half; with one half mixing with the Control siRNA and the other with the SETX siRNA
   5. Add 100ul of siRNA mix per well to 1ml of plates
7. Incubate at 37C overnight
8. After ~16hr, change media
9. After ~40hr, proceed to immunofluorescence protocol

Immunofluorescence Staining:

1. Washed cells with 1ml 1x PBS once
2. Fixed with 300ul 4% PFA for 10min at RT with rocking 
3. Washed 2x with 1ml 1x PBS
4. Permeabilized with 300ul of 0.25% Triton-X100 in PBS for 10min at RT with rocking
5. Washed briefly 2x with 1ml 1x PBS
6. Optional Steps: Enzymatic Pre-digestion to remove signal
   1. Dilute RNase III 1:150 in low salt buffer and incubated with 200ul/well at 37C for ~1 hr
   2. After digestion, washed briefly with 1ml PBST 2x then 1ml 1x PBS once
7. Blocked with 300ul/well 3% BSA in PBS at RT for 30min with rotation
   1. Filter sterilize the 3% BSA solution
8. Incubated samples with 200ul/well of primary diluted in 3% BSA/PBS at 4C o/n with rocking
   1. 1:200 dilution for J2 antibody
9. Washed 2x with 1ml PBST for 5min with rotation at RT, then 1x with 1ml PBS for 5min at RT with rotation
10. Incubated in secondary antibody at RT for 1hr with rotation
    1. Goat anti-Mouse 594: 1:1000
    2. DAPI: 1:1000
    3. HCS: 0.3ul
11. Washed 3x with 1ml PBS for 5min at RT with rotation
12. Mounted coverslips using Prolong Gold
13. Imaged the next day using a Zeiss LSM800 confocal microscope equipped with an AxioImager.Z2 microscope using a 63×/1.40 NA differential interference contrast oil objective (Zeiss) with oil immersion.

Low Salt Buffer                                                                         PBST Wash Buffer

50mM Tris-HCl, pH 7.6                                                              1x PBS

75mM KCl                                                                                0.05% Tween-20

3mM MgCl₂

0.1% BSA

Cat. No.’s:

Shortcut RNase III             à NEB; M0245S

J2 Antibody                        à Scicon; 10010200

594 secondary                   à Invitrogen; A32742

HCS Cell Mask Deep Red à Thermo Fisher Scientific; H32721