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=FISSEQ on BA8 Sections with PA gel + Acrydite/BS(PEG)9= *Used worst section from [[Matt:LabNotes/2016-1-20|1/19/16 BA8 patient 1568 from Yun]] *These coverslips have only Vectabond (-NH2 primary amines) and no Bind-Silane *Gel mix and protocol is same as [[Matt:LabNotes/2016-2-18|this successful experiment]] **Except half the A/B mix to make 5% *4% Formaldehyde is two weeks old and has been sitting at RT **From <http://swehsc.pharmacy.arizona.edu/sites/swehsc.pharmacy.arizona.edu/files/docs/ci/formaldehyde.pdf> **"We recommend that 10% buffered formalin solutions be used no longer than 3 months after they were initially mixed. The solution should be clear, colorless, with no precipitate and the pH should not be below 6.5." ==Protocol== ===Day 1=== #Prepare plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate #*EtOH and UV sterilize #Used 4% PFA from 6/15/2016 #Take out worst BA8 section that is left from -80C and dry on 50C hot plate for 3min #Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C #Wash twice with cold 1X SSPE by submerging #Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT #Wash with cold nf-H2O three times '''and check for degradation''' #Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C #*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O #Wash with nf-1X PBS three times '''and check for degradation''' #Aspirate any liquid and place a glass slide with 120um spacer over the coverslip with tissue #*Secure in plastic jig #Add 100ul gel casting mix '''filtered and degassed''' #*5% gel with 500uM Acrydite-Amine linker and 5mM BS(PEG)9 #Seal in plastic bag and vacuum out air before filling with argon #Let sit at RT for 30min #Aspirate non-polymerized gel and wash once with 1X PBS #Attach coverslip to bottom of petri dish #*COVERSLIP CRACKED DIAGONALLY WHEN CLEANING AWAY GEL #*NEED TO BE MORE CAREFUL WHEN HANDLING *Continued by testing for swelling #Put each half of cracked coverslip in petri dish with 1M Tris and put in 37C overnight ===Results=== [[File:20160630_GelTissueSwellTest_1.JPG|450px]] [[File:20160630_GelTissueSwellTest_2.JPG|450px]] *Next morning the gels were still stuck to glass *No significant swelling **Vertical length of gel was ~17mm when cast (no image but that is the length of the mold) and is ~17mm after overnight incubation in Tris **Suggests that the gel is covalently bound to glass ===Conclusion=== *Making gel with Acrydite/Amine linker and BS(PEG)9 is viable solution to covalently bond polyacrylamide gel to Vectabond treated glass and tissue sections
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