Matt:LabNotes/2015-10-29: Difference between revisions

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>Mzcai
(Created page with "=DARTFISH on BA8 Sections= *Now I know the regions that have been degrading the most are the gray matter with cortical layers **Need this to compare with Gwen's tilescan image...")
 
>Mzcai
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#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
#Wash with cold nf-H2O three times '''and check for degradation'''
#Wash with nf-1X PBS three times '''and check for degradation'''
#Prepare Reverse Transcription Mix '''on ice''' and add
#Prepare Reverse Transcription Mix '''on ice''' and add
{| {{table}}
{| {{table}}

Revision as of 06:14, 30 October 2015

DARTFISH on BA8 Sections

  • Now I know the regions that have been degrading the most are the gray matter with cortical layers
    • Need this to compare with Gwen's tilescan images
  • Try 90sec 0.01% pepsin incubation and pay close attention to gray matter region for degradation

Protocol

Day 1

  1. Prepare 1 plastic culture dish with 12x17mm hole, tweezers, large plastic dish, and 50C hot plate
    • EtOH and UV sterilize
  2. Use 40ml 4% PFA at RT made by Dan on same day
    • 10ml 16% PFA from ampule + 4ml 10X PBS + 26ml nuclease-free H2O?
  3. Take out last BA8 section (~1 month ago) from -80C and incubate 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
  7. Add 0.25% TX-100 in 2XSSPE and incubate 10min at RT
  8. Wash with cold nf-H2O three times and check for degradation
  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 and check for degradation
  11. Prepare Reverse Transcription Mix on ice and add
Components Volume
H2O 159
10X M-MuLV Buffer 20
25mM dNTP 2
4mM aa-dUTP 2
100uM N9 5
RNase Inhibitor 2
M-MuLV RTase 10
Total 200
  1. Incubate 10min at 4C and then ~18hr at 37C