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=Mouse Brain FISSEQ test= *Repeat [[Matt:LabNotes/2016-8-18|yesterday's]] experiment but with way less pepsin ==Previous mouse brain FISSEQ tries== [[Hosuk:LabNotes/2014-5-19|2nd try]]<br> [[Hosuk:LabNotes/2014-5-25|3rd try]]<br> [[Hosuk:LabNotes/2014-5-30|4th try]]<br> [[Hosuk:LabNotes/2014-6-18|7th try]]<br> [[Hosuk:LabNotes/2014-6-27|9th try]]<br> [[Hosuk:LabNotes/2014-7-1|4X BF Image of mouse brain sections from Yun]]<br> ==Sample Info== *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== ===Summary=== *Try without hydrogel, hopefully no degradation *Try 3 samples with 3 different pepsin incubation times #1 min #5 min #10 min ===Day 1=== #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|450px]] #Prepare 400ul Reverse Transcription Mix '''on ice''' and add ~133ul to each dish {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''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=== #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=== #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]]
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