Matt:LabNotes/2015-10-7: Difference between revisions
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=FISSEQ on Mouse Embryo Test= | =FISSEQ on Mouse Embryo Test= | ||
*Try 2 samples: | *Try 2 samples: slide5 (20um thickness) and slide6 (25um thickness) | ||
==Embryo Section Info== | |||
#E7.5#3 slide1 is 16um (8 sections beginning of embryo + placenta) on the coverslip there is written A1, B1, C1, D1 to help for the orientation. | |||
#E7.5#3 slide2 is 20um (8 sections beginning of embryo + placenta) on the coverslip there is written A2, B2, C2, D2 to help for the orientation. | |||
#E7.5#3 slide3 is 25um (7 sections beginning of embryo + placenta) on the coverslip there is written A3, B3, C3, D3 to help for the orientation. | |||
#E7.5#3 slide4 is 16um (7 sections mainly embryo) on the coverslip there is written A4, B4, C4, D4 to help for the orientation. | |||
#E7.5#3 slide5 is 20um (7 sections middle embryo) on the coverslip there is written A5, B5, C5, D5 to help for the orientation. | |||
#E7.5#3 slide6 is 25um (7 sections middle embryo) on the coverslip there is written A6, B6, C6, D6 to help for the orientation. | |||
#E7.5#3 slide7 is 16um (10 sections middle embryo) on the coverslip there is written A7, B7, C7, D7 to help for the orientation. | |||
#E7.5#3 slide8 is 20um (12 sections middle/end embryo) on the coverslip there is written A8, B8, C8, D8 to help for the orientation. | |||
#E7.5#3 slide9 is 25um (6 sections end embryo+ adult liver) on the coverslip there is written A9, B9, C9, D9 to help for the orientation. | |||
==Protocol== | ==Protocol== | ||
*For CircLigase reaction use [[Matt:JustinLabNotes/2015-10-7 | MnCl2 made by Justin]] and 5M Betaine from Sigma | |||
===Day 1=== | ===Day 1=== | ||
#Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O | #Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O | ||
#Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole | #Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole | ||
#*Use Double-sided tape: Adhesives Research IS-7876-35 | #*Use Double-sided tape: Adhesives Research IS-8458-19 (Hosuk originally mistakenly told me IS-7876-35) | ||
#Wash tissue section twice using cold nf-H2O for 5min each | #Wash tissue section twice using cold nf-H2O for 5min each | ||
#Add 0.25% TX-100 in nf-H2O for 5min at RT | #Add 0.25% TX-100 in nf-H2O for 5min at RT | ||
Line 35: | Line 47: | ||
#Incubate 10min at 4C and then ~18hr at 37C | #Incubate 10min at 4C and then ~18hr at 37C | ||
#*Parafilm each dish and then put in plastic bag with wet tissue | #*Parafilm each dish and then put in plastic bag with wet tissue | ||
===Day 2=== | |||
#Wash with 1X PBS twice | |||
#*Dishes had small leak so had to seal with gap-filling glue | |||
#Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Quench with 1M Tris for 30min at RT | |||
#Wash with 1X PBS twice | |||
#RNA Removal | |||
#*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C | |||
#Wash with H2O twice | |||
#Add 200ul CircLigaseII mix and incuabate 3hr at 60C | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| DEPC-H2O||640 | |||
|- | |||
| CircLigase Buffer 10X||100 | |||
|- | |||
| MnCl2 50mM||50 | |||
|- | |||
| Betaine 5M|| 200 | |||
|- | |||
| CircLigase II (100U/ul)||10 | |||
|- | |||
| Total||1000 | |||
|} | |||
#Wash with 1X PBS twice | |||
#Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C | |||
#Wash with 2X SSC + 30% formamide for 10min at 60C | |||
#wash with 2X SSC, 1X SSC, 1X PBS once each | |||
#Add 200ul RCA mix and incubate for 15hr at 30C | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| H2O||174 | |||
|- | |||
| 10X Phi29 Buffer||20 | |||
|- | |||
| 25mM dNTP||2 | |||
|- | |||
| 4mM aa-dUTP||2 | |||
|- | |||
| Phi 29 (low conc)||2 | |||
|- | |||
| Total||200 | |||
|} | |||
===Day 3=== | |||
#Wash with 1X PBS once | |||
#Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Quench with 1M Tris for 30min at RT | |||
#Wash with 1X PBS twice | |||
#Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT | |||
#Wash with 2X SSC twice | |||
#Image with Confocal | |||
==Image Results== | |||
===slide 5 20um=== | |||
*Section 1 10X | |||
[[File:Cropped_BF_20151009_E7.5_a5_20um_10X_Section1.jpg]] | |||
*Section 2 10X | |||
[[File:Cropped_BF_20151009_E7.5_a5_20um_10X_Section2.jpg]] | |||
*Section 1 20X | |||
[[File:MAX_20151009_E7.5_a5_20um_FISSEQ_20X_Cy3.jpg|450px]] [[File:Merge_MAXandBF_20151009_E7.5_a5_20um_FISSEQ_20X_Cy3.jpg|450px]] | |||
*Section 1 63X | |||
[[File:MAX_20151009_E7.5_a5_20um_FISSEQ_63X_Cy3.jpg|450px]] [[File:Merge_MAXandBF_20151009_E7.5_a5_20um_FISSEQ_63X_Cy3.jpg|450px]] | |||
*Section 2 had no fluorescence in Cy3 channel for no apparent reason | |||
===slide 6 25um=== | |||
*10X | |||
[[File:Cropped_BF_20151009_E7.5_a6_25um_10X.jpg]] | |||
*63X | |||
[[File:MAX_20151009_E7.5_a6_25um_FISSEQ_63X_Pos2_Cy3.jpg|450px]] [[File:Merge_MAXandBF_20151009_E7.5_a6_25um_FISSEQ_63X_Pos2_Cy3.jpg|450px]] | |||
=FISSEQ on BA8 for Harvard= | =FISSEQ on BA8 for Harvard= | ||
Line 46: | Line 134: | ||
#Submerge coverslip1 in 5ml 4% PFA in 6 well culture plate for 15min at 37C | #Submerge coverslip1 in 5ml 4% PFA in 6 well culture plate for 15min at 37C | ||
#Submerge glass slides in 35ml 4%PFA in 50ml tube for 15min at 37C | #Submerge glass slides in 35ml 4%PFA in 50ml tube for 15min at 37C | ||
#Wash with 2X SSPE by submerging | #Wash twice with 2X SSPE by submerging | ||
#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 | ||
#Aspirate any liquid and attach gaskets to glass slides | #Aspirate any liquid and attach gaskets to glass slides | ||
Line 75: | Line 163: | ||
| Total||600 | | Total||600 | ||
|} | |} | ||
#Incubate 10min at 4C and then ~ | #Incubate 10min at 4C and then ~13hr at 37C | ||
#*Put slide in | #*Put slide in 50ml tube with wet towel and para film around cap | ||
#*Parafilm dish and put in humidified bag with mouse E7.5 | #*Parafilm dish and put in humidified bag with mouse E7.5 | ||
===Day 2=== | |||
#Wash with 1X PBS twice | |||
#Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Quench with 1M Tris for 30min at RT | |||
#Wash with 1X PBS twice | |||
#RNA Removal | |||
#*Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C | |||
#Wash with H2O twice | |||
#Add 200ul CircLigaseII mix and incuabate 3hr at 60C | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| DEPC-H2O||640 | |||
|- | |||
| CircLigase Buffer 10X||100 | |||
|- | |||
| MnCl2 50mM||50 | |||
|- | |||
| Betaine 5M|| 200 | |||
|- | |||
| CircLigase II (100U/ul)||10 | |||
|- | |||
| Total||1000 | |||
|} | |||
#Wash with 1X PBS twice | |||
#Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C | |||
#Wash with 2X SSC + 30% formamide for 10min at 60C | |||
#wash with 2X SSC, 1X SSC, 1X PBS once each | |||
#Add 200ul RCA mix and incubate for 15hr at 30C | |||
{| {{table}} | |||
| align="center" style="background:#f0f0f0;"|'''Component''' | |||
| align="center" style="background:#f0f0f0;"|'''Volume''' | |||
|- | |||
| H2O||174 | |||
|- | |||
| 10X Phi29 Buffer||20 | |||
|- | |||
| 25mM dNTP||2 | |||
|- | |||
| 4mM aa-dUTP||2 | |||
|- | |||
| Phi 29 (low conc)||2 | |||
|- | |||
| Total||200 | |||
|} | |||
===Day 3=== | |||
#Wash with 1X PBS once | |||
#Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT | |||
#Wash with 1X PBS twice | |||
#Quench with 1M Tris for 30min at RT | |||
#Wash with 1X PBS twice | |||
#Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT | |||
#Wash with 2X SSC twice | |||
#Image with Confocal | |||
==Image Results== | |||
===63X=== | |||
[[File:MAX_20151009_BA8_FISSEQ_63X_Pos1.jpg|550px]]<br> | |||
[[File:Merge_MAXandBF_20151009_BA8_FISSEQ_63X_Pos1.jpg|550px]] | |||
=Conclusion and Take aways= | |||
*FISSEQ rolonies in BA8 less than usual | |||
**Guessing "home made" MnCl2 from dry crystal not good enough, will order 50mM solution from Trevigen | |||
***CircLigase II epicentre kit is on back order so can't wait for that | |||
*Significant degradation of mouse embryo sections | |||
**Try pipetting solutions to side of tissue instead of dropping on top | |||
**Try fixing tissue again after permeabilization | |||
*Based on z-start and z-end positions there does not seem to be a difference in tissue thickness between 20um section and 25um section | |||
**Both measured to be ~15um | |||
**Is there some maximum tissue thickness that can withstand degradation? | |||
**Maybe tissue thickness isn't being measured but just where rolonies are | |||
*Weird spots in mouse embryo tissue (looks like precipitate?) | |||
**They do not appear in BA8 tissue | |||
**They do not overlap with rolonies |
Latest revision as of 23:04, 14 December 2015
FISSEQ on Mouse Embryo Test[edit]
- Try 2 samples: slide5 (20um thickness) and slide6 (25um thickness)
Embryo Section Info[edit]
- E7.5#3 slide1 is 16um (8 sections beginning of embryo + placenta) on the coverslip there is written A1, B1, C1, D1 to help for the orientation.
- E7.5#3 slide2 is 20um (8 sections beginning of embryo + placenta) on the coverslip there is written A2, B2, C2, D2 to help for the orientation.
- E7.5#3 slide3 is 25um (7 sections beginning of embryo + placenta) on the coverslip there is written A3, B3, C3, D3 to help for the orientation.
- E7.5#3 slide4 is 16um (7 sections mainly embryo) on the coverslip there is written A4, B4, C4, D4 to help for the orientation.
- E7.5#3 slide5 is 20um (7 sections middle embryo) on the coverslip there is written A5, B5, C5, D5 to help for the orientation.
- E7.5#3 slide6 is 25um (7 sections middle embryo) on the coverslip there is written A6, B6, C6, D6 to help for the orientation.
- E7.5#3 slide7 is 16um (10 sections middle embryo) on the coverslip there is written A7, B7, C7, D7 to help for the orientation.
- E7.5#3 slide8 is 20um (12 sections middle/end embryo) on the coverslip there is written A8, B8, C8, D8 to help for the orientation.
- E7.5#3 slide9 is 25um (6 sections end embryo+ adult liver) on the coverslip there is written A9, B9, C9, D9 to help for the orientation.
Protocol[edit]
- For CircLigase reaction use MnCl2 made by Justin and 5M Betaine from Sigma
Day 1[edit]
- Sterilize culture dishes and tweezers with EtOH, RNaseZap, and then rinse with nf-H2O
- Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 16mm diameter hole
- Use Double-sided tape: Adhesives Research IS-8458-19 (Hosuk originally mistakenly told me IS-7876-35)
- Wash tissue section twice using cold nf-H2O for 5min each
- Add 0.25% TX-100 in nf-H2O for 5min at RT
- Wash with cold nf-H2O twice
- Add 200ul 0.01% pepsin in 0.01N HCl (2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O) for 10min at RT
- Wash with 2ml of cold nf-PBS three times
- Prepare 2X Reverse Transcription Mix on ice
Components | Volume |
H2O | 157 |
10X M-MuLV Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
100uM FISSEQ_RT | 5 |
RNase Inhibitor | 2 |
M-MuLV RTase | 10 |
Total | 200 |
- Incubate 10min at 4C and then ~18hr at 37C
- Parafilm each dish and then put in plastic bag with wet tissue
Day 2[edit]
- Wash with 1X PBS twice
- Dishes had small leak so had to seal with gap-filling glue
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
- Wash with H2O twice
- Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component | Volume |
DEPC-H2O | 640 |
CircLigase Buffer 10X | 100 |
MnCl2 50mM | 50 |
Betaine 5M | 200 |
CircLigase II (100U/ul) | 10 |
Total | 1000 |
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Wash with 2X SSC + 30% formamide for 10min at 60C
- wash with 2X SSC, 1X SSC, 1X PBS once each
- Add 200ul RCA mix and incubate for 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice
- Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT
- Wash with 2X SSC twice
- Image with Confocal
Image Results[edit]
slide 5 20um[edit]
- Section 1 10X
File:Cropped BF 20151009 E7.5 a5 20um 10X Section1.jpg
- Section 2 10X
File:Cropped BF 20151009 E7.5 a5 20um 10X Section2.jpg
- Section 1 20X
File:MAX 20151009 E7.5 a5 20um FISSEQ 20X Cy3.jpg File:Merge MAXandBF 20151009 E7.5 a5 20um FISSEQ 20X Cy3.jpg
- Section 1 63X
File:MAX 20151009 E7.5 a5 20um FISSEQ 63X Cy3.jpg File:Merge MAXandBF 20151009 E7.5 a5 20um FISSEQ 63X Cy3.jpg
- Section 2 had no fluorescence in Cy3 channel for no apparent reason
slide 6 25um[edit]
- 10X
File:Cropped BF 20151009 E7.5 a6 25um 10X.jpg
- 63X
File:MAX 20151009 E7.5 a6 25um FISSEQ 63X Pos2 Cy3.jpg File:Merge MAXandBF 20151009 E7.5 a6 25um FISSEQ 63X Pos2 Cy3.jpg
FISSEQ on BA8 for Harvard[edit]
- 1 slide with 2 sections and 1 coverslip
Day 1[edit]
- Prepare 1 plastic culture dishes with hole, tweezers, large plastic dish, slide gasket, and 50C hot plate
- EtOH and RNaseZap sterilize and rinse with nf-H2O
- Made 40ml 4% PFA at RT
- 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nf-H2O
- Take out BA8 section (1 glass slide1 and 1 coverslip1) from -80C and incubate on 50C hot plate for 3min
- Submerge coverslip1 in 5ml 4% PFA in 6 well culture plate for 15min at 37C
- Submerge glass slides in 35ml 4%PFA in 50ml tube for 15min at 37C
- Wash twice with 2X SSPE by submerging
- Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
- Aspirate any liquid and attach gaskets to glass slides
- Add 0.25% TX-100 in 2XSSPE and incubate 5min at RT
- Wash with nuclease-free H2O three times
- Add 0.01% Pepsin in 0.01N HCl and incubate for 5min at 37C
- 2ul 1%Pepsin + 1ul 2N HCl + 197ul H2O
- Wash with nuclease-free PBS three times
- Prepare 3X Reverse Transcription Mix on ice
Components | Volume |
H2O | 477 |
10X M-MuLV Buffer | 60 |
25mM dNTP | 6 |
4mM aa-dUTP | 6 |
100uM N9 or 100uM FISSEQ_RT | 15 |
RNase Inhibitor | 6 |
M-MuLV RTase | 30 |
Total | 600 |
- Incubate 10min at 4C and then ~13hr at 37C
- Put slide in 50ml tube with wet towel and para film around cap
- Parafilm dish and put in humidified bag with mouse E7.5
Day 2[edit]
- Wash with 1X PBS twice
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- Add 200ul RNase Mix (100ul RNase H 10X Buffer + 10ul Riboshredder + 50ul RNase H + 840ul H2O) and incubate 1hr at 37C
- Wash with H2O twice
- Add 200ul CircLigaseII mix and incuabate 3hr at 60C
Component | Volume |
DEPC-H2O | 640 |
CircLigase Buffer 10X | 100 |
MnCl2 50mM | 50 |
Betaine 5M | 200 |
CircLigase II (100U/ul) | 10 |
Total | 1000 |
- Wash with 1X PBS twice
- Add 200ul 0.5uM FISSEQ_RCA (5ul 100uM FISSEQ_RCA + 995ul 2X SSC + 30% formamide) and incubate 1hr at 60C
- Wash with 2X SSC + 30% formamide for 10min at 60C
- wash with 2X SSC, 1X SSC, 1X PBS once each
- Add 200ul RCA mix and incubate for 15hr at 30C
Component | Volume |
H2O | 174 |
10X Phi29 Buffer | 20 |
25mM dNTP | 2 |
4mM aa-dUTP | 2 |
Phi 29 (low conc) | 2 |
Total | 200 |
Day 3[edit]
- Wash with 1X PBS once
- Add 200ul BS(PEG)9 (20ul BS(PEG)9 stock + 980ul 1X PBS) and incubate 1hr at RT
- Wash with 1X PBS twice
- Quench with 1M Tris for 30min at RT
- Wash with 1X PBS twice
- Add 200ul 75C preheated 0.5uM FISSEQ_Adpt (Cy3) in 2X SSC + 30% formamide and incubate 10min at RT
- Wash with 2X SSC twice
- Image with Confocal
Image Results[edit]
63X[edit]
File:MAX 20151009 BA8 FISSEQ 63X Pos1.jpg
File:Merge MAXandBF 20151009 BA8 FISSEQ 63X Pos1.jpg
Conclusion and Take aways[edit]
- FISSEQ rolonies in BA8 less than usual
- Guessing "home made" MnCl2 from dry crystal not good enough, will order 50mM solution from Trevigen
- CircLigase II epicentre kit is on back order so can't wait for that
- Guessing "home made" MnCl2 from dry crystal not good enough, will order 50mM solution from Trevigen
- Significant degradation of mouse embryo sections
- Try pipetting solutions to side of tissue instead of dropping on top
- Try fixing tissue again after permeabilization
- Based on z-start and z-end positions there does not seem to be a difference in tissue thickness between 20um section and 25um section
- Both measured to be ~15um
- Is there some maximum tissue thickness that can withstand degradation?
- Maybe tissue thickness isn't being measured but just where rolonies are
- Weird spots in mouse embryo tissue (looks like precipitate?)
- They do not appear in BA8 tissue
- They do not overlap with rolonies