Immunofluorescence and analyses of PB number and ER-PB colocalization

Immunofluorescence

1. U-2 OS cells were seeded at 0.8x10⁵ cells/ml on fibronection-coated coverslips.
2. 30 hours after plating, cells were fixed with 37°C fixative solution (4% paraformaldehyde, 4% sucrose in PBS) for 10 min. 
3. Fixed cells were washed once with 2mL phosphate buffered saline (PBS).
4. Cells were permeabilized with 0.1% Triton-X100 for 5 minutes.
5. Cells were blocked with 5% normal donkey serum in PBS for 30 minutes. 
6. Labeling of PBs and ER was achieved by incubating cells overnight at 4°C with 1:200 Edc3 mouse monoclonal (Santa Cruz sc-271806) and 1:200 Calreticulin polyclonal rabbit (Abcam - ab2907) antibodies in blocking serum. 
7. Cells were then washed with PBS for 5 minutes. Three washes.
8. Primary antibodies from step 6 were fluorescently labeled with 1:400 donkey–anti-mouse 488 and 1:400 donkey–anti-rabbit 594 secondary antibodies (Invitrogen A32766 and A32754) for 1 hour. 
9. Cells were then washed with PBS for 5 minutes. Three washes.
10. Nuclei were labeled with 1:2000 Hoecsht (10mg/mL – Thermofisher H3570)
11. Cells were then washed with PBS for 5 minutes. Three washes.
12. Coverslips were mounted on microscope slides using Prolong glass resin. 

Imaging

Using a 100x objective, Z-stack images of cells from each condition were captured on spinning disc confocal microscope (should work with other similar scopes with high enough resolution to resolve ER tubule network). Images were from each experimental replicate were captured within the same day under identical conditions with respect to laser intensities and exposures. These images can be used to calculate both parameters below (PB number and ER-PB colocalization).

Quantifying P-body number

*Only the P-body Z-stack is needed.

1. Image > Stacks > Z-project > “Max Intensity”. 
2. Obtain the maximum fluorescence intensity of the cytosol by drawing a box in the cytosolic space excluding P-bodies. Analyze > Measure to obtain the maximum fluorescence intensity (x). This is especially critical for cells that do not have P-bodies, which prevents thresholding calculations from segmenting the dilute phase.
3. Use "Process-->Math-->Subtract..." and enter x to subtract background. 
4. Segmentation of PBs was accomplished by Otsu thresholding in ImageJ. Image > Adjust > Auto Threshold > Select “Yen” and check “white objects on black background”.
5. Use Analyze > Analyze particles to quantify the number of P-bodies in the image. 

Quantifying ER tubule and P-body colocalization using Manders coefficient

* The level of colocalization between PBs and ER tubules was accomplished by selecting region of interest (ROIs) that contained at least one PB and resolvable ER tubules. ROIs were necessary because the ER network is too dense to resolve in regions within the cell, such as the microtubule organizing center.

1. ~8-10 micron² ROIs with the criteria of less than 50% ER coverage area and one P-body off-center were selected. 
2. Segmentation of PBs was accomplished by Otsu thresholding in ImageJ. Image > Adjust > Auto Threshold > Select “Otsu” and check “white objects on black background”.
3. Segmentation of the ER was accomplished by manual thresholding owing to the broad range of ER labeling intensities throughout the cell and between ROIs. Image > Adjust > Manual Threshold. 
4. Calculate Mander’s correlation coefficient using ImageJ. Analyze > Colocalization > coloc2 > check “Mander’s Correlation”. This coefficient = M1_PB.
5. Rotate the thresholded ER image 90 degrees (from step 3).
6. Calculate Mander’s correlation coefficient using ImageJ. Analyze > Colocalization > coloc2 > check “Mander’s Correlation”. This coefficient = M1₉₀.

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