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*[[Hosuk:Lab_Notes|LabNote]] ===Human Brain Tissue : Mild Pepsin condition with BA8=== *Samples from Yun **S1: BA8 **S2: BA8 ====Procedure==== *12/17 *#Take Tissue glass slide from -80C, and put it in a well of sterilized 6 well plate to 50C (hot plate) for 3min. (To prevent RNase activity) *#Add 4% formamide on the glass, incubate for 30min at room temp. *#Wash tissue with '''2x SSPE''' twice for 5min each *#Aspirate '''2x SSPE''', and wipe solution around the edge of the glass *#'''Apply adhesive to glass, place glass to MatTek dish, wait for 15min for complete dry''' *#Wash with '''2x SSPE''' twice (add DEPC-PBS on a well, aspirate) *#Add 0.25% Triton X-100 in '''2x SSPE''' for 15 min *#Pepsin incubation - '''S1:0.01% for 5 min each, S2:0.01% for 15 min each''' *#Wash with DEPC-H2O three times *#Add RT mix (Hexamer, -a-dUTP) *#*DEPC-H2O 161uL *#*M-MuLV Buffer 20uL *#*25mM dNTP 2uL *#*100uM random hexamer RT Primer 5uL *#*Rnase Inhibitor 2uL *#*M-MuLV reverse transcriptase 10 uL *#Incubate the specimen with the reaction mixture for 10 min at 4°C, then transfer to 37°C for overnight. *12/18 *#Skip BS(PEG)9 step, Wash with PBS once *#Add RNase H mix, incubate for 1hr. at 37C *#*RNase H mix : 2 ul Riboshredder (Epicentre)+ 10 ul RNase H(Enzymatics) in 20 ul RNase H buffer(Enzymatics) + 168 uL H2O *#Wash with nucleaus-free H2O twice *#Add CircLigase II mix, incubate at 60C for 2hr. (CircLigase II was too small volume left ~1uL) *#*H2O 128uL *#*Buffer 20uL *#*50mM MnCl2 10uL *#*5M Betaine 40uL *#*CircLigaseII 2uL *#RCA Primer, final concentration : 0.5uM in 200uL (2x SSC + 30% Formamide) at 60C for 1hr. *#1uL of 100uM RCA Primer + 199uL 2X SSC with 30% Formamide *#Wash with 2XSSC twice, and 1X SSC twice *#Prepare RCA reaction mix *#*H2O 172uL *#*Buffer 20uL *#*25mM dNTP 2uL *#*2mM a-dUTP 4uL *#*Phi29(low conc.) 2uL *#Add RCA mix, incubate at 30C overnight *12/19 *#Wash with PBS once *#Prepare 4uL of BS(PEG)9 in 196uL PBS *#add 200uL BS(PEG)9 mix, incubate for 1hr at RT *#Wash with PBS once, add Tris pH 8.0, incubate for 30min *#Wash with PBS once *#Prepare detection Probe : Final Conc. 1uM --> 2uL of 100uM Cy3-Adapt + 198uL (2xSSC + 30% Formamide) *#Preheat detection Probe at 80C for 5min *#Add to sample and incubate at RT for 10min *#Wash two times for 1 minute each with 1 mL 1X PBS ====Result==== *Imaging condition **20x, 4k, Pinhole = 4, Laser = 4, PMT = 620 (580 for S4), 20x 4K *Result : **S1, 0.01% Pepsin for 5 min showed similar good result than before(12/12 sample) **S2 0.01% Pepsin for 15 min was not good. **Tissue of S1 was remained well, but S2 tissue were degraded. *Next Plan... *Let’s try TX-100 for 10min, and 0.01% Pepsin for 5min *I need to wait for a new CircLigase II! (it's back ordered now...) ====Result Images : prelim scanning==== *20x, 4k * S1, # = 11654 *[[File:BA8_2014-12-19_S1_Pos3_Images_2.png|1200px]]
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