Brietschopf & Suchanek 1 of 2

Modified IN SITU Hybridization

IN SITU Hybridization

Modified protocol from Brietschopf and Suchanek, “Detection of mRNA on paraffin-embedded material of the central nervous system with DIG-labeled RNA probes,” Boehringer Nonradioactive In Situ Hybridization Application Manual, p. 136.

These parameters are designed around 7-10 um paraffin sections fixed in 85% EtOH, 10% formaldehyde (from 37% stock), 5% glacial acetic acid (Wilkinson, 1992).

Note: All prehyb solutions made with 0.1% DEPC-treated Milli-Q water.

In situ Hybridization on sections:

Day 1:

  1. Dewax in xylene 2X 15 min each
  2. Rehydrate 100%, 95%, 70%, 50% EtOH, H2O, PBS 2 min each
  3. Postfix in freshly prepared 4% paraformaldehyde in PBS 20 min RT
  4. 3X TBS wash (50 mM Tris, pH 7.5, 150 mM NaCl) à 5, 10, 10 min RT
  5. 200 mM HCL 10 min RT (4 mL HCl in 246 mL H2O)
  6. 3X TBS à 5, 10, 10 min RT
  7. Acetic anhydride (Prepare immediately before use) 10 min RT, with vigorous stirring. (25 mL 1M Tris, pH 8.0, 224 mL H2O, 1.25 mL acetic anhydride)
  8. 3X TBS wash à 5, 10, 10 min RT
  9. Proteinase K 10 ug/ml in TE (10 mM Tris, pH 8.0, 1mM EDTA) 15 min RT. Prepare as 125 ul of 20mg/ml Proteinase K stock in 250 ml TE
  10. Wash 3X with 4˚C TBS à 5, 10, 10 min
  11. Remove excess TBS with Chemwipe and apply probe (10 ng in 60 ul hyb buffer per slide containing 4 or 5 sections). Hyb buffer prepared as 2X SSC, 10 % dextran sulfate, 0.01% yeast tRNA, 0.02% SDS, 50% formamide).
  12. Place coverslipped slides on heating block prewarmed to 95˚C for 4 min
  13. Remove from heat and seal slides with rubber cement
  14. Hyb O/N @ 55˚C.

Day 2:

  1. Remove rubber cement and hold slides in 2X SSC until through with all
  2. Wash 3X 20 min in 1:1 formamide:2X SSC at 55˚C
  3. Wash 2X 15 min in 1X SSC at RT
  4. Equilibrate in TBS (3X 5 min each)
  5. PAP pen and block 1 hr RT in Roche blocking agent (dilute 10X block stock in 100 mM maleic acid, 150 mM NaCl, pH 7.5) containing 10% fetal calf serum (FCS) and 1% sheep serum
  6. dilute anti-DIG 1:1000 in block containing 10% FCS and incubate sections 1 hr RT
  7. Wash 4X in TBS (5, 10, 10, 10 min)
  8. Equilibrate sections in AP buffer2 min (100 mM Tris, pH 9.5, 50 mM MgCl2, 100 mM NaCl)
  9. Place Roche NBT/BCIP substrate solution. Filter solution through 0.22 um syringe filter before placing on sections.
  10. Place at 4˚C in covered, humidified container. Next a.m., usually no development yet, replaced solution and kept covered at RT during the day, checking regularly for development. If needed to go longer than 24 hours, placed at 4˚C O/N again and remove the next a.m. Development times between 24-46 hours worked well.

01/02/07