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=BA8 Section with Fiducial Beads= *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== ===Fixation and Permeabilization of BA8=== #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 <!--#*Still had leak so used gap-filling adhesive 200 to seal--> #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=== #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== *Using Gain of 550 (what I regularly use for decoding 488 channel) *Pos1 [[File:MAX_BA8_FiducialBeads_Try2_Pos1.jpg|650px]] *Pos2 [[File:MAX_BA8_FiducialBeads_Try2_Pos2.jpg|650px]] *Pos3 (area that is more degraded) [[File:MAX_BA8_FiducialBeads_Try2_Pos3.jpg|650px]]
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