Matt:LabNotes/2015-6-30: Difference between revisions
Jump to navigation
Jump to search
>Mzcai (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 m (→Results) |
||
(7 intermediate revisions by the same user not shown) | |||
Line 20: | Line 20: | ||
**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
- 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
- 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[edit]
- Using CUX2-488 KIT-594 KIT-647
- 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[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