TITLE: Immunofluorescent Imaging and Analysis
AUTHORS: Dan Tudorica

[DESCRIPTION]
Following treatment with antibodies/genetic methods of introducing fluorescent tags

[STEPS]
SECTION: Preparation of chambered slides
1. Seed cells on 8-well chambered slide to be 80% confluent on day of analysis.

SECTION: Preparation of chambered slides
2. Wash cells once with PBS, then fix for 15 minutes using 4% PFA diluted in PBS. Wash cells once more with PBS.
SECTION: Preparation of chambered slides
3. Permeabilize cells for an additional 15 minutes using a 1x stock of SLO prepared in PBS + 10 mM TCEP. Prepare 1x stock according to the protocol in this article (https://doi.org/10.7554/eLife.20378)
SECTION: Preparation of chambered slides
4. Remove SLO, wash cells with PBS once
SECTION: Preparation of chambered slides
5. Incubate permeabilized cells in 2% BSA in PBS for 1 H at RT to block
SECTION: Preparation of chambered slides
6. Replace blocking solution with blocking solution + IFA competent primary antibody. For this study, used a 1:200 dilution of ab302494 anti pS72 Rab7 Rb antibody. Incubate at RT for 1 H
SECTION: Preparation of chambered slides
7. Wash cells 3 x 5 min with PBS to remove primary antibody
SECTION: Preparation of chambered slides
8. Replace PBS with a solution of fluorescently labelled secondary antibody diluted in 2% BSA in PBS. In this study, used ThermoFisher antibody A11008, Goat anti Rabbit Alexa 488. Incubate for 1 H in the dark.
SECTION: Preparation of chambered slides
9. Wash cells with 3 x 5 min of PBS, then leave cells sitting under PBS. Image immediately.
SECTION: Imaging
10. Prepare Confocal microscope with 60x immersion oil objective. Place slides on stage, and find focus.
SECTION: Imaging
11. Adjust laser power, pinhole, and gain for optimal brightness and resolution. 
SECTION: Imaging
12. Capture each fluorescent channel individually in order to minimize risk of bleedthrough
SECTION: Imaging
13. In order to test for bleedthrough, check to make sure that when excitatory laser for a particular channel is dialed down to an intensity of 0, image is completely dark.
SECTION: Imaging
14. Acquire images and export to FIJI for analysis
SECTION: Colocalization analysis
15. Merge fluorescent channels into a single stack for colocalization analysis
SECTION: Colocalization analysis
16. For each channel, measure the max brightness of a known dark region of the image. For this study, I used the nucleus of a cell as an expected dark. Use FIJI's math>subtract command to subtract this value from each pixel in the image. 
SECTION: Colocalization analysis
17. In each image, define ROIs around target cells. If a transient transfection was performed, cells should be expressing a moderate amount of the target protein.
SECTION: Colocalization analysis
18. Use the Coloc 2i plugin to calculate the Mander's colocalization coefficient with a Costes' threshold regression.
SECTION: Colocalization analysis
19. Perform this analysis on at least 20 different cells imaged from different parts of the well. The average of these cells constitute a single biological replicate. Repeat this experiment from the start several more times, and then perform statistics on aggregated experimental averages.