Matt:LabNotes/2015-8-18: Difference between revisions

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=BA8 Section DARTFISH suppv2 with Fiducial Beads=
=BA8 Section DARTFISH suppv2 with Fiducial Beads=
*Similar to [[Matt:LabNotes/2015-8-4]] but with suppressor oligos and 5 cycle decoding
*Repeat [[Matt:LabNotes/2015-8-17]] but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation


*3-4 month old BA8 tissue section
*3-4 month old BA8 tissue section
*suppv2 design
*suppv2 design
*PFA frozen from [[Matt:LabNotes/2015-8-4]]
*Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
*Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers


*Try only 5min incubation with TX-500 to see if that decreases tissue degradation
*Try only 5min incubation with TX-500 to see if that decreases tissue degradation
==DARTFISH on BA8==
==DARTFISH on BA8==
#Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
#Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
#*EtOH sterilize and UV
#*EtOH sterilize and UV
#Thaw 5ml 4% PFA at RT
#Made 40ml 4% PFA at RT
#*10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
#Take out BA8 section from -80C and incubate on 50C hot plate for 3min
#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
#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
#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
#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 5min at RT
#Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
#*New solution made by Justin
#*New solution made by Justin
#Wash with nuclease-free H2O three times
#Wash with nuclease-free H2O three times
#Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
#Add 0.01% Pepsin in 0.1N HCl and incubate for 5min at 37C
#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
#*2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
#*There was '''significant''' degradation after this step, only a sliver of tissue left
#Wash with nuclease-free PBS three times
#Wash with nuclease-free PBS three times
#Prepare Reverse Transcription Mix '''on ice'''
#Prepare Reverse Transcription Mix '''on ice'''
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| Total||200
| Total||200
|}
|}
#Incubate 10min at 4C and then ~18hr at 37C
#Incubate 10min at 4C and then ~18hr at 37C<!--
#Wash with 1X PBS once
#Wash with 1X PBS once
#Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT
#Add 300ul BS(PEG)9 (6ul BS(PEG)9 + 294ul 1X PBS) and incubate 1hr at RT

Revision as of 22:14, 18 August 2015

BA8 Section DARTFISH suppv2 with Fiducial Beads

  • Repeat Matt:LabNotes/2015-8-17 but with fresh PFA and 0.01N HCl (instead of 0.1N during pepsin step) to see if that helps with tissue degradation
  • 3-4 month old BA8 tissue section
  • suppv2 design
  • Use FluoSpheres Amine-Modified Microspheres 0.2um (505/515) as fiducial markers
  • Try only 5min incubation with TX-500 to see if that decreases tissue degradation

DARTFISH on BA8

  1. Prepare plastic culture dish with hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH sterilize and UV
  2. Made 40ml 4% PFA at RT
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O
  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 5min at RT
    • New solution made by Justin
  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 + 1ul 2N HCl + 197ul H2O
  10. Wash with nuclease-free PBS three times
  11. Prepare Reverse Transcription Mix on ice
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
2mM aa-dUTP 4
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C