Matt:LabNotes/2016-8-18: Difference between revisions

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>Mzcai
(Created page with "=Mouse Brain FISSEQ test= *Repeat yesterday's experiment but with way less pepsin ==Previous mouse brain FISSEQ tries== [[Hosuk:LabNotes/2014-5-19...")
 
>Mzcai
 
(7 intermediate revisions by the same user not shown)
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#Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
#Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
#Wash with cold nf-H2O three times '''and check for degradation'''
#Wash with cold nf-H2O three times '''and check for degradation'''
#Add 100ul 0.1% Pepsin in 0.1N HCl and incubate for X min at 37C
#Add 100ul 0.01% Pepsin in 0.1N HCl and incubate for X min at 37C
#*30ul 1%Pepsin + 15ul 2N HCl + 255ul H2O
#*3ul 1%Pepsin + 15ul 2N HCl + 282ul H2O
#*Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 10min
#*Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 5min
#*Sample 2 was already pretty degraded when I took it out so I also took sample 3 out around 5min mark
#Wash with nf-1X PBS three times '''and check for degradation'''
#Wash with nf-1X PBS three times '''and check for degradation'''
#*Sample 2 and 3 could see tissue sloughing off with each wash, doubt anything will be left after RT overnight
[[File:20160818_MouseBrain_Section1.JPG|450px]]
#Prepare 400ul Reverse Transcription Mix '''on ice''' and add ~133ul to each dish
#Prepare 400ul Reverse Transcription Mix '''on ice''' and add ~133ul to each dish
{| {{table}}
{| {{table}}
Line 62: Line 65:
|}
|}
#Incubate 10min at 4C and then ~15hr at 37C
#Incubate 10min at 4C and then ~15hr at 37C
<!--
 
===Day 2===
===Day 2===
#Wash with 1X PBS once
#Wash with 1X PBS once
#*Sample 2 and 3 are too degraded, only continued with sample 1
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
#Wash with 1X PBS twice
#Wash with 1X PBS twice
Line 72: Line 76:
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
#*Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
#Wash with nf-H2O twice
#Wash with nf-H2O twice
-->
#Add CircLigase mix and incubate 4hr at 60C
#*H2O - 128ul
#*CircLigase Buffer 10X - 20ul
#*MnCl2 50mM - 10ul
#*Betaine 5M - 40ul
#*CircLigase II 100U/ul - 2ul
#Wash with 1X PBS twice
#Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
#Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
#Prepare RCA reaction mix '''on ice'''
#*H2O - 174ul
#*Phi29 Buffer 10X - 20ul
#*dNTP 25mM - 2ul
#*aa-dUTP 4mM - 2ul
#*Phi29 DNA polymerase 100U/ul - 2ul
#Add RCA mix and incubate at 30C overnight (~15hrs)
===Day 3===
#Wash with 1X PBS once
#Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
#Wash with 1X PBS twice
#Add 1M Tris pH 8.0 and incubate 30min at RT
#Wash with 1X PBS twice
 
==Imaging==
===8-23-2016===
====Full_3D_Tilescan====
*20X objective to image WHOLE section in 110 FOVs (10 rows 11 columns)
**Software couldn't stitch, have to do it in Fiji/ImageJ
====Full_2D_Tilescan====
*Still using 20X objective
*Chose a single z-plane so it would be faster and better chance of software stitching it together
**Uneven lighting for each FOV made stitching ugly
[[File:CroppedFull_2D_Tilescan_Stitched_Cy3.jpg|450px]]
[[File:CroppedFull_2D_Tilescan_Stitched_BF.jpg|450px]]
 
====63x_example====
*Try using 63x objective to take single FOV image
**Position was 6 columns from left, 3 columns from bottom of image
*Image not shown here because better images with DRAQ5 below
 
===8-25-2016===
*Tilescan 3D with 63X
**Randomly chose an area in the middle
====TileScan_MouseBrain_FISSEQ_63x_Cy3====
*Once Cy3 and BF only
====TileScan_MouseBrain_FISSEQ_63x_Cy3_DRAQ5====
*Once Cy3 DRAQ5 and BF
*Added 5uM DRAQ5 in PBS and let sit for 15min with no washing
*DRAQ5 "quenches" Cy3 fluorescence so have to use Cy3 from previous image
 
[[File:TileScan_MouseBrain_FISSEQ_63x_Cy3_DRAQ5_Stitched.jpg|800px]]<br>
[[File:TileScan_MouseBrain_FISSEQ_63x_BF_Stitched.jpg|800px]]

Latest revision as of 19:07, 4 September 2016

Mouse Brain FISSEQ test[edit]

Previous mouse brain FISSEQ tries[edit]

2nd try
3rd try
4th try
7th try
9th try
4X BF Image of mouse brain sections from Yun

Sample Info[edit]

  • Received 10 x 10um thick sections of mouse brain on Vectabond and Bind-Silane treated coverglass
    • Each section is half of coronal section
  • Fresh frozen tissue, cryostat into 10um sections on vectabond coverslips, dry for 30min at -20C in cryostat chamber
  • Box up and freeze at -80C
  • Coverslip attached to glass slide by a drop of frozen water

FISSEQ Protocol[edit]

Summary[edit]

  • Try without hydrogel, hopefully no degradation
  • Try 3 samples with 3 different pepsin incubation times
  1. 1 min
  2. 5 min
  3. 10 min

Day 1[edit]

  1. Prepare 3 plastic culture dishes with 10mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Make 4% PFA in 1X PBS
    • 10ml 16% PFA + 4ml 10X PBS + 26ml H2O (made fresh)
  3. Take out mouse brain section from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Attach coverslip to bottom of petri dish with ARcare 90106 double sided adhesive
  7. Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
  8. Wash with cold nf-H2O three times and check for degradation
  9. Add 100ul 0.01% Pepsin in 0.1N HCl and incubate for X min at 37C
    • 3ul 1%Pepsin + 15ul 2N HCl + 282ul H2O
    • Sample 1 = 1min, Sample 2 = 5min, Sample 3 = 5min
    • Sample 2 was already pretty degraded when I took it out so I also took sample 3 out around 5min mark
  10. Wash with nf-1X PBS three times and check for degradation
    • Sample 2 and 3 could see tissue sloughing off with each wash, doubt anything will be left after RT overnight

File:20160818 MouseBrain Section1.JPG

  1. Prepare 400ul Reverse Transcription Mix on ice and add ~133ul to each dish
Components Volume
H2O 159
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM RT primer 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~15hr at 37C

Day 2[edit]

  1. Wash with 1X PBS once
    • Sample 2 and 3 are too degraded, only continued with sample 1
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice
  6. RNA Removal
    • Add 200ul RNase Mix (20ul RNase H 10X Buffer + 2ul Riboshredder + 10ul RNase H + 168ul H2O) and incubate 1hr at 37C
  7. Wash with nf-H2O twice
  8. Add CircLigase mix and incubate 4hr at 60C
    • H2O - 128ul
    • CircLigase Buffer 10X - 20ul
    • MnCl2 50mM - 10ul
    • Betaine 5M - 40ul
    • CircLigase II 100U/ul - 2ul
  9. Wash with 1X PBS twice
  10. Add 200ul (1ul 200uM FISSEQ_RCA + 199ul 2X SSC + 30% formamide) and incubate 1hr at 60C
  11. Aspirate and wash with 2X SSC, then 1X SSC, and finally 1X PBS
  12. Prepare RCA reaction mix on ice
    • H2O - 174ul
    • Phi29 Buffer 10X - 20ul
    • dNTP 25mM - 2ul
    • aa-dUTP 4mM - 2ul
    • Phi29 DNA polymerase 100U/ul - 2ul
  13. Add RCA mix and incubate at 30C overnight (~15hrs)

Day 3[edit]

  1. Wash with 1X PBS once
  2. Add 200ul cold BS(PEG)9 (4ul stock BS(PEG)9 + 196ul 1X PBS) and incubate 1hr at RT
  3. Wash with 1X PBS twice
  4. Add 1M Tris pH 8.0 and incubate 30min at RT
  5. Wash with 1X PBS twice

Imaging[edit]

8-23-2016[edit]

Full_3D_Tilescan[edit]

  • 20X objective to image WHOLE section in 110 FOVs (10 rows 11 columns)
    • Software couldn't stitch, have to do it in Fiji/ImageJ

Full_2D_Tilescan[edit]

  • Still using 20X objective
  • Chose a single z-plane so it would be faster and better chance of software stitching it together
    • Uneven lighting for each FOV made stitching ugly

File:CroppedFull 2D Tilescan Stitched Cy3.jpg File:CroppedFull 2D Tilescan Stitched BF.jpg

63x_example[edit]

  • Try using 63x objective to take single FOV image
    • Position was 6 columns from left, 3 columns from bottom of image
  • Image not shown here because better images with DRAQ5 below

8-25-2016[edit]

  • Tilescan 3D with 63X
    • Randomly chose an area in the middle

TileScan_MouseBrain_FISSEQ_63x_Cy3[edit]

  • Once Cy3 and BF only

TileScan_MouseBrain_FISSEQ_63x_Cy3_DRAQ5[edit]

  • Once Cy3 DRAQ5 and BF
  • Added 5uM DRAQ5 in PBS and let sit for 15min with no washing
  • DRAQ5 "quenches" Cy3 fluorescence so have to use Cy3 from previous image

File:TileScan MouseBrain FISSEQ 63x Cy3 DRAQ5 Stitched.jpg
File:TileScan MouseBrain FISSEQ 63x BF Stitched.jpg