Matt:LabNotes/2015-8-13

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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]

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Thaw 5ml 4% PFA at RT
  3. Take out BA8 section from -80C and incubate on 50C hot plate for 3min
  4. Submerge section in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash with 2X SSPE by submerging in 2 clean wells of culture plate with 5ml SSPE
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with nuclease-free H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nuclease-free PBS three times

Add Beads[edit]

  1. Wash with 10mM HEPES
  2. 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
  3. Sonicate for 3 minutes with microtip probe
    • Power = 3, 3sec on 2sec off
  4. Add to sample and incubate 5min at RT
  5. Wash with 10mM HEPES once
  6. Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 20mM HEPES) and incubate at RT for 1hr
  7. Wash with 1X PBS twice
  8. 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)

File:MAX BA8 FiducialBeads Try2 Pos3.jpg