Matt:LabNotes/2016-1-27: Difference between revisions

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>Mzcai
(Created page with "=FISSEQ on Mouse Embryo Test 2= * Previous test using sections of fixed embryo *This time try fresh frozen mouse embryo sections ==Embryo Sectio...")
 
>Mzcai
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==Protocol==
==Protocol==
*Previously did 0.01% Pepsin for 10min at RT and had too much tissue loss
*Try 0.01% Pepsin for 1-2min at 37C


===Day 1===
===Day 1===
#Prepare 2 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
#Prepare 2 plastic culture dish with 18mm hole, tweezers, large plastic dish, and 50C hot plate
#*EtOH and UV sterilize
#*EtOH and UV sterilize
#Use 1 week old 4% PFA at RT
#Use 1 week old 4% PFA at RT
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##Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
##Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
#Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
#Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
#Wash with cold nf-H2O three times '''and check for degradation'''
#*Both dishes leaked and required glue
#*Slightly more stripes in gray matter
#Wash with cold nf-H2O three times
#Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
#Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
#*2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O

Revision as of 02:57, 28 January 2016

FISSEQ on Mouse Embryo Test 2

Embryo Section Info

Protocol

Day 1

  1. Prepare 2 plastic culture dish with 18mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Use 1 week old 4% PFA at RT
  3. Take out fresh frozen mouse embryo sections from -80C and dry on 50C hot plate for 3min
  4. Submerge coverslip in 5ml 4% PFA in 6 well culture plate for 15min at 37C
  5. Wash twice with cold 1X SSPE by submerging
  6. Aspirate any liquid and attach glass to bottom of culture dish with double-sided adhesive
    1. Attach 22mm x 22mm glass coverslips (with mounted sections) to bottom of culture dishes with 18mm diameter hole (16mm adhesive hole)
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
    • Both dishes leaked and required glue
  8. Wash with cold nf-H2O three times
  9. Add 0.01% Pepsin in 0.1N HCl and incubate for 90sec at 37C
    • 2ul 1%Pepsin + 10ul 2N HCl + 188ul H2O
  10. Wash with nf-1X PBS three times
  11. Prepare Reverse Transcription Mix on ice and add
Components Volume
H2O 157
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM FISSEQ_RT 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C
    • Parafilm each dish and then put in plastic bag with wet tissue