Matt:LabNotes/2015-8-13
Jump to navigation
Jump to search
BA8 Section with Fiducial Beads[edit]
- Last time 1:500 dilution of beads: Matt:LabNotes/2015-8-4
- This time try 1:2000 dilution
- Skip DARTFISH protrocol and add beads right after permeabilization
- Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
Procedure[edit]
Fixation and Permeabilization of BA8[edit]
- Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
- EtOH sterilize and UV
- Thaw 5ml 4% PFA at RT
- Made on Matt:LabNotes/2015-8-4
- Take out BA8 section from -80C and incubate on 50C hot plate for 3min
- Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
- Wash with nuclease-free PBS three times
Add Beads[edit]
- Wash with 10mM HEPES
- Dilute beads 1:2000 in 10mM HEPES (1ul stock beads + 999ul H2O + 1000ul 20mM HEPES pH 7.3)
- Make 500ul aliquots of 1:2000 diluted beads and store in dcProbe box in 4C
- Sonicate for 3 minutes with microtip probe
- Power = 3, 3sec on 2sec off
- Add to sample and incubate 5min at RT
- Wash with 10mM HEPES once
- Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate at RT for 30min
Confocal Imaging Results[edit]
- Using Gain of 550 (what I regularly use for decoding 488 channel)
- Pos1
File:MAX BA8 FiducialBeads Try2 Pos1.jpg
- Pos2
File:MAX BA8 FiducialBeads Try2 Pos2.jpg
- Pos3 (area that is more degraded)