Matt:LabNotes/2016-8-18
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Mouse Brain FISSEQ test[edit]
- Repeat yesterday's experiment but with way less pepsin
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 min
- 5 min
- 10 min
Day 1[edit]
- Prepare 3 plastic culture dishes with 10mm hole, tweezers, large plastic dish, and 50C hot plate
- EtOH and UV sterilize
- Make 4% PFA in 1X PBS
- 10ml 16% PFA + 4ml 10X PBS + 26ml H2O (made fresh)
- Take out mouse brain section from -80C and dry on 50C hot plate for 3min
- Submerge coverslip in 5ml 4% PFA in petri dish for 15min at 37C
- Wash twice with cold 1X SSPE by submerging
- Attach coverslip to bottom of petri dish with ARcare 90106 double sided adhesive
- Add 100ul 0.25% TX-100 in 2XSSPE and incubate 15min at RT
- Wash with cold nf-H2O three times and check for degradation
- 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
- 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
- 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 |
- Incubate 10min at 4C and then ~15hr at 37C
Day 2[edit]
- 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
- Wash with 1X PBS twice
- Add 1M Tris pH 8.0 and incubate 30min at RT
- Wash with 1X PBS twice
- RNA Removal
- 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
- 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[edit]
- 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[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