Admin: Created page with "= Notes = For imaging, use a space ship with the inner surface painted flat black The dilution for the primary Fab' must be worked out for each antibody, and after each di..."

2021-02-01T19:56:45Z

Created page with "= Notes = *For imaging, use a space ship with the inner surface painted flat black *The dilution for the primary Fab' must be worked out for each antibody, and after each di..."

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= Notes =

*For imaging, use a space ship with the inner surface painted flat black
*The dilution for the primary Fab' must be worked out for each antibody, and after each digest.
*Image capture should be done in phenol-red free media such as HBSS or PBS. Inhibitors should be added to imaging media unless inhibitor autofluorescence is an issue
*Antibody addition can be done at RT or on ice. Ice may alter cytoskeleton, particularity microtubules.
*Q-dots must be added on ice to prevent endocytosis of dots.

= Protocol =

===== Cell Preparation: =====

#Serum starve cells for 30min-24hrs in DMEM or HPMI, 37oC.
#Prepare antibodies & inhibitors: 
#*Dilute antibodies into HPMI + 4% donkey serum, as per table below 
#*Prepare 2 volumes of inhibitors in HPMI, and 1 volume inhibitors in HBSS 
#Treat cells with inhibitors for 10-30min at 37oC
#Block cells for 10min with 4% donkey serum, on ice or RT.
#Without washing, add primary antibody, 10min on ice or RT.
#Wash 2-3X with HPMI.
#Add secondary antibody, 10min on ice or RT.
#Wash 2-3X with HPMI. 
#*If using Q-dots wash with HBSS or PBS (no biotin!) 
#Add tertiary antibody, 10min on ice or RT. 
#*If using Q-dots, add 1:10,000 Q-dots, ice cold, 4min 
#*Incubate with ice-cold HPMI (has biotin) for 2-3min. 
#Wash 2-3X with HPMI. 
#Add inhibitors to cells, in HPMI, keep on ice or at RT until imaging.

===== Imaging: =====

#30 min prior to imaging turn on Bert and both cameras & warm PBS+inhibitors to 37oC
#*Camera power supplies are on top shelf. 
#Set EPI to max, no lasers are needed
#Start velocity in 3DM mode: 
#*Open new file 
#*Start Video Preview: 
#**Select lower of two camera under Video -> Source 
#**Video -> Show Saturation 
#*Set Capture to Maximum Speed 
#*Open Acquisition Panel 
#**Capture wCy3 channel only 
#**30-50 timepoints (do not use points per second) 
#**Max Sample Protection 
#*Set wCy3 (white Cy3 button) exposure to 100ms, sensitivity to max, gain to best Signal-to-Noise ratio 
#**If using Q-dots use Q-dot channel (button beside wCy3 button) 
#Check that ND filter at back of scope is 100%. If not, ND's are stored in the wooden box behind the chair. 
#Set path to Eye and focus on sample, find cell using DIC
#Set path to Bottom Port, using lower of the port-switching buttons
#Focus cell using DIC, focus on edge of cell, avoiding cell body; capture a snapshot
#Switch to wCy3, focus using autolevels, record time series as soon as dots are focused 
#*Click mouse off of air table, or on frame of air table, to avoid motion 
#Capture 5-10 cells per condition

===== Image Processing: =====

#Move all DIC images into a sub-folder
#Move all time sequences into a second fold; then duplicate this folder
#Crop the duplicates using the freehand crop tool and Action -> Crop to Selection
#Split image volume into individual images within a folder, using Image -> Split Volume
#Export as TIFF, each time lapse into its own folder 
#*Export as regular TIFF 
#*Number, pad with 3 zeros 
#*Append library or item name, use underscores to connect name parts 
#Transfer to server

Single Particle Tracking - Revision history

Admin: Created page with "= Notes = *For imaging, use a space ship with the inner surface painted flat black *The dilution for the primary Fab' must be worked out for each antibody, and after each di..."

Admin: Created page with "= Notes = For imaging, use a space ship with the inner surface painted flat black The dilution for the primary Fab' must be worked out for each antibody, and after each di..."