Daniel:Notebook/RNAFISH/2014-9-3: Difference between revisions

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(Created page with "=Trial Run (Started 08/29/2014)= Back to Calendar ==Post-Hybidization== #Remove rubber cement together wit...")
 
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#Remove rubber cement together with the chamber using a pair of fine forceps
#Remove rubber cement together with the chamber using a pair of fine forceps
#Wash slides in 2C SSC at 37°C, 3 × 15 min (preferably with shaking) to remove hybridization mixture
#Wash slides in 2X SSC at 37°C, 3 × 15 min (preferably with shaking) to remove hybridization mixture
#Perform stringency washes in 0.1X SSC at 60°C, 2 × 5 min to remove nonspecific hybrids
#Perform stringency washes in 0.1X SSC at 60°C, 2 × 5 min to remove nonspecific hybrids
#Equilibrate in 2X SSC for 2 min
#Equilibrate in 2X SSC for 2 min

Revision as of 16:02, 3 September 2014

Trial Run (Started 08/29/2014)

Back to Calendar

Post-Hybidization

  1. Remove rubber cement together with the chamber using a pair of fine forceps
  2. Wash slides in 2X SSC at 37°C, 3 × 15 min (preferably with shaking) to remove hybridization mixture
  3. Perform stringency washes in 0.1X SSC at 60°C, 2 × 5 min to remove nonspecific hybrids
  4. Equilibrate in 2X SSC for 2 min
  5. Add DAPI, let sit for at least 30 minutes
  6. Seal the coverslip with nail polish; let sit 30 minutes
    1. Sections become fragile after the FISH; therefore, strong pressure on the coverslip should be avoided
    2. e.g., pressing the coverslip with the microscope objective when focusing often completely destroys the sample