Matt:LabNotes/2015-6-30: Difference between revisions

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(Created page with "=Motor Neurons RNA FISH with cooled CCD= *Photometric QUANTEM Camera **Cooled to -30C **100X objective *Ex/Em Filters available 450px *Alexa488 **49...")
 
>Mzcai
 
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**647/665 (Cy5)
**647/665 (Cy5)
**Filter 4/Filter 6-7
**Filter 4/Filter 6-7
*[[Matt:LabNotes/2015-6-8 | First Try]]
*[[Matt:LabNotes/2015-6-18 | Second Try]]
*[[Matt:LabNotes/2015-6-15 | Probe dye coupling]]
*[[Matt:LabNotes/2015-6-18 | Second batch iPS derived motor neurons from Yeo Lab (Sebastian)]]
**Permeabilized/stored in 70% EtOH at 4C (2 weeks before RNA FISH experiment)
===RNA FISH Procedure===
*Using [[Matt:LabNotes/2015-6-15#Dye_Coupling | CUX2-488 KIT-594 KIT-647]]
*[[Daniel:Notebook/RNAFISH/2015-3-4 | Dan's best practice FISH protocol]]
*[[Daniel:Protocols/TissueFISH#Linnarsson_Lab_Protocol | Linnarsson complete FISH protocol]]
*Sample 1 labeled: "RNAFISH1 6.30.2015"
**488
*Sample 2 labeled: "RNAFISH2 6.30.2015"
**594
*Sample 3 labeled: "RNAFISH3 6.30.2015"
**647
#Prepare 10ml Wash Buffer and let sit at RT
#Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
#*Aliquots made by Dan
#Prepare hybridization reaction
#*Hybridization Buffer 100ul
#*Probe 5ul
#Wash the cells with 1ml of PBS (RNase free) 2 times
#Add 2ml Wash Buffer and let sit 10min at RT
#Aspirate
#Add 105ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)
#Prepare 15ml Wash Buffer
#Warm up 12ml Wash Buffer and 3ml Wash Buffer with DAPI to 37C
#Rinse with 2ml Wash Buffer
#Add 2ml Wash Buffer and incubate 30min at 37C
#Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C
#Wash with 2X SSC (warmed to 30C) twice
#Add 2X SSC
====Buffer Prep====
*Wash Buffer
**20X SSC 5mL
**Formamide 5mL
**RNase free H2O 40ml
*Wash Buffer with DAPI
**Wash Buffer 10ml
**DAPI (100ug/ml) 50ul
***Stored in brown opaque tube in 4C
*Hybridization Buffer
**RNAse free water 5.3 mL
**SSC 20X 1 mL
**Dextran sulfate 2 mL
**Formamide 1 mL
**E coli tRNA 500 uL
**RVC 200 mM (warm to 37) 100 uL
**BSA 50 mg/mL 40 uL
===Results===
*Only CUX2 (488) probes worked
**My guess is CUX2 is most highly expressed in these cells
**To confirm next time I can use CUX2 probes labeled with 2 different dyes
***Odd probes labeled one color and even probes labeled the other, should see colocalization of 2 colors
====Raw Images====
*RNAFISH1_Pos1
**Exposure only 1sec vs 5sec for other images
[[File:2_w1Alexa488.jpg|350px]] [[File:2_w2DIC.jpg|350px]]
*RNAFISH1_Pos2
[[File:3_w1Alexa488.jpg|350px]] [[File:3_w2DIC.jpg|350px]]
*RNAFISH1_Pos3
[[File:5_w1Alexa488.jpg|350px]] [[File:5_w2DIC.jpg|350px]]
*RNAFISH1_Pos4
[[File:7_w1Alexa488.jpg|350px]] [[File:7_w2DIC.jpg|350px]]
*RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''')
[[File:Dish3_5_w1Alexa488.jpg|350px]] [[File:Dish3_5_w2DIC.jpg|350px]]
*RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''')
[[File:Dish3_6_w1Alexa488.jpg|350px]] [[File:Dish3_6_w2DIC.jpg|350px]]
====StarSearch====
*Use [http://rajlab.seas.upenn.edu/StarSearch/launch.html StarSearch from Raj Lab]
**Doesn't work in Chrome browser (use IE)
**Uses a histogram of pixel intensities and a threshold to determine how many "stars"
*RNAFISH1_Pos2
[[File:3_w1Alexa488_StarSearch.png]]
*RNAFISH1_Pos3
[[File:5_w1Alexa488_StarSearch.png]]
*RNAFISH1_Pos4
[[File:7_w1Alexa488_StarSearch.png]]
*RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''')
**Can't process image, something about StarSearch algorithm makes image almost all white/saturated
*RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for '''Negative Control''')
**Identified 6 spots but you can tell they're not real distinct spots
[[File:Dish3_6_w1Alexa488_StarSearch.png]]

Latest revision as of 16:56, 3 July 2015

Motor Neurons RNA FISH with cooled CCD[edit]

  • Photometric QUANTEM Camera
    • Cooled to -30C
    • 100X objective
  • Ex/Em Filters available

File:IMG 0517.JPG

  • Alexa488
    • 495/519 (FITC equivalent)
    • Filter 5/Filter 2
  • Alexa564
    • 556/573 (tetramethylrhodamine)
    • No filters available
  • Alexa594
    • 590/617 (Texas Red)
    • Filter 8/Filter 5
  • Alexa647
    • 647/665 (Cy5)
    • Filter 4/Filter 6-7

RNA FISH Procedure[edit]

  • Sample 1 labeled: "RNAFISH1 6.30.2015"
    • 488
  • Sample 2 labeled: "RNAFISH2 6.30.2015"
    • 594
  • Sample 3 labeled: "RNAFISH3 6.30.2015"
    • 647
  1. Prepare 10ml Wash Buffer and let sit at RT
  2. Thaw 500ul aliquot Hybridization Buffer and warm up to 37C
    • Aliquots made by Dan
  3. Prepare hybridization reaction
    • Hybridization Buffer 100ul
    • Probe 5ul
  4. Wash the cells with 1ml of PBS (RNase free) 2 times
  5. Add 2ml Wash Buffer and let sit 10min at RT
  6. Aspirate
  7. Add 105ul hybridization solution to each sample and incubate overnight at 37C (~23hrs)
  8. Prepare 15ml Wash Buffer
  9. Warm up 12ml Wash Buffer and 3ml Wash Buffer with DAPI to 37C
  10. Rinse with 2ml Wash Buffer
  11. Add 2ml Wash Buffer and incubate 30min at 37C
  12. Replace with 2ml Wash Buffer with DAPI and incubate 30min at 30C
  13. Wash with 2X SSC (warmed to 30C) twice
  14. Add 2X SSC

Buffer Prep[edit]

  • Wash Buffer
    • 20X SSC 5mL
    • Formamide 5mL
    • RNase free H2O 40ml
  • Wash Buffer with DAPI
    • Wash Buffer 10ml
    • DAPI (100ug/ml) 50ul
      • Stored in brown opaque tube in 4C
  • Hybridization Buffer
    • RNAse free water 5.3 mL
    • SSC 20X 1 mL
    • Dextran sulfate 2 mL
    • Formamide 1 mL
    • E coli tRNA 500 uL
    • RVC 200 mM (warm to 37) 100 uL
    • BSA 50 mg/mL 40 uL

Results[edit]

  • Only CUX2 (488) probes worked
    • My guess is CUX2 is most highly expressed in these cells
    • To confirm next time I can use CUX2 probes labeled with 2 different dyes
      • Odd probes labeled one color and even probes labeled the other, should see colocalization of 2 colors

Raw Images[edit]

  • RNAFISH1_Pos1
    • Exposure only 1sec vs 5sec for other images

File:2 w1Alexa488.jpg File:2 w2DIC.jpg

  • RNAFISH1_Pos2

File:3 w1Alexa488.jpg File:3 w2DIC.jpg

  • RNAFISH1_Pos3

File:5 w1Alexa488.jpg File:5 w2DIC.jpg

  • RNAFISH1_Pos4

File:7 w1Alexa488.jpg File:7 w2DIC.jpg

  • RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)

File:Dish3 5 w1Alexa488.jpg File:Dish3 5 w2DIC.jpg

  • RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)

File:Dish3 6 w1Alexa488.jpg File:Dish3 6 w2DIC.jpg

StarSearch[edit]

  • Use StarSearch from Raj Lab
    • Doesn't work in Chrome browser (use IE)
    • Uses a histogram of pixel intensities and a threshold to determine how many "stars"
  • RNAFISH1_Pos2

File:3 w1Alexa488 StarSearch.png

  • RNAFISH1_Pos3

File:5 w1Alexa488 StarSearch.png

  • RNAFISH1_Pos4

File:7 w1Alexa488 StarSearch.png

  • RNAFISH3_Pos1 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)
    • Can't process image, something about StarSearch algorithm makes image almost all white/saturated
  • RNAFISH3_Pos2 with 488 excitation (use exact same microscope settings as RNAFISH1 for Negative Control)
    • Identified 6 spots but you can tell they're not real distinct spots

File:Dish3 6 w1Alexa488 StarSearch.png