Double-label in situ hybridization (whole mounts)

Protocol for Whole Mount in situ Hybridization with TSA & Fast Red or NBT/BCIP

Barthel, L.K. and P.A. Raymond (2000). In situ hybridization studies of retinal neurons. In Vertebrate Phototransduction and the Visual Cycle, K. Palczewski, ed., Methods in Enzymology, vol. 316, Part B, J.N. Abelson and M.I. Simon, eds., Academic Press, Orlando, FL pp. 579-590.

Day 1:
Prehybridization: RNase Free

  1. Rinse in 100% MetOH for 5 min, RT
  2. Rinse in 50:50 MetOH and Xylene for 5 min, RT
  3. Rinse in 100% Xylene for 30 min, RT
  4. Rinse in 100% MetOH for 30 min, RT
  5. Rehydrate tissue at RT
    a. 90% MetOH for 5 min
    b. 70% MetOH for 5 min
    c. 50% MetOH for 5 min
  6. Rinse in 1X PBS/0.1% Tween 2 times, 15 min each at RT
  7. Make Proteinase K buffer and prewarm to 37°C (see next page)
  8. Add 25 µl Proteinase K to 37°C buffer, add to tissue for 15 min in 37°C water bath
  9. Rinse embryos for 10-15 sec in 1X PBS/0.1% Tween at RT
  10. Re-fixation of tissue:
    a. Remove 1X PBS/0.1% Tween and add 4% paraformaldehyde in PBS for 20 min at RT
    b. Remove fixative and wash with PBS/Tween for 5 min 2 times
  11. Rinse in 0.1 M TEA for 3 minutes at RT
  12. Incubate tissue in Acetic Anhydride/TEA solution for 10 min at RT (see next page)
  13. Rinse for 30 min in 1X PBS/0.1% Tween
  14. Prewarm Prehybridization and hybridization solutions to 56°C (see next page)
  15. Add 500 µl of Prehybridization solution to each tube, incubate for 1-2 hours at 56°C
  16. Prepare DIG and/or FL probes (2 µg/2µl each):
    a. Add calculated amount of DEPC H2O to tubes containing probes & heat at 65°C for 10 min
    (500µl – 436µl + probe volume= amount of DEPC to add).
    If double labeling, add 1/2 the calculated amount of DEPC to each tube.
    b. Place immediately on ice
  17. Remove Prehybridization solution from tubes with tissue, add 436µl of hybridization solution, then add DEPC/probe(s)
  18. Hybridize overnight at 56°C
TEA solution
0.93 g Triethanolamine
50 ml DEPC H2O
Proteinase K buffer
2.5 ml 1 M Tris, pH 8.0
2.5 ml 0.5 M EDTA
20 ml DEPC H2O
(25 µl of Proteinase K will be
added to this)
Acetic Anhydride Sol’n
32.5 µl Acetic Anhydride
12.5 ml TEA
Prehybridization Sol’n 50 ml
3.6 ml TEN sol’n
25 ml 100 % Formamide
10 ml 50% Dextran Sulfate
5 ml 10% RMB blocker
6.4 ml DEPC H2O
Store at -20oC
Hybridization sol’n 43.6 ml
3.6 ml TEN sol’n
25 ml 100 % Formamide
10 ml 50% Dextran Sulfate
5 ml 10% RMB blocker
6.4 ml DEPC H2O
NO water—add later with probe
Store at -20°C
10X PBS solution
2.76 g NaH2PO4 x H2O (monobasic)
11.36 g Na2HPO4 (dibasic)
87.6 g NaCl
1.87 g KCl
Bring up to 1 liter with DEPC H2O
TEN Solution
5 ml of 1.0M Tris-HCl, pH 7.5
30 ml of 5M NaCl
1 ml of 0.5M EDTA

Day 2:
Posthybridization: No longer use RNase free procedures

  1. Prewarm the following solutions:
    a. 10 ml 50% formamide/ 2X SSC to 65°C
    b. 10 ml 2X SSC at 37°C
    c. 10 ml RNase buffer/ 0.1% Tween sol’n to 37°C
    d. 10 ml RNase buffer/0.1% Tween sol’n to 65°C
  2. Rinse briefly in 2X SSC, RT
  3. Wash 1 hour in 50% formamide/ 2X SSC in 65°C water bath
  4. Wash in 37°C 2X SSC 3 times for 10 min each in 37°C water bath
  5. RNase A:
    a. Add 20 µl of RNase A (10 mg/ml) to 10 ml of 37°C RNase buffer/0.1% Tween
    b. incubate tissue with RNase buffer/ 0.1% Tween sol’n for 1 hr at 37°C
  6. Wash in 65°C RNase buffer/0.1% Tween without RNase A for 30 min
  7. Wash 2 times for 15 min each in 2X SSC at 37°C
  8. Wash in 1X PBS/ 0.1% Triton for 15 min at RT
  9. Transfer tissue to new silconized tube
  10. Block with Maleate/Triton/RMB blocker solution for 2 hours
  11. Incubate in a-DIG and/or a-FL antibodies in 1X Maleate/0.05% Triton/1% RMB blocker solution overnight at 4°C (see concentrations below)

1X Maleate/0.05% Triton/1.0% RMB blocker solution
2 ml of 5X Maleate
5 µl Trition
1 ml of 10% RMB Blocker
make 3 ml aliquots and freeze at -200°C
When ready to use, add 7 ml of milli-Q water to 3 ml aliquot of blocking

Antibody / 1X Maleate/0.25% Triton/1.0 % RMB blocker solution
anti-DIG-AP 1:5000
anti-DIG-POD 1:100
anti-FL-AP 1:100
anti-FL-POD 1:100

RNase Buffer (pH 7.5)

0.5 M NaCl / 10 mM Tris/ 1mM EDTA
29.23 NaCl
10 ml 1M Tris pH 7.5
2 ml 0.5 M EDTA
bring up to 1L with DEPC H2O
RNase Buffer/ 0.1% Tween
100 ml RNase Buffer
100 µl Tween


**Begin with a 1X Maleate buffer/ 0.1% Triton wash, 3 times, 10 min each**
Proceed onto a color reaction: (TSA is usually done last in double-labeling)

TSA-Biotin System:
(uses -POD tagged probes, no solid precipitate, FITC fluorophore, visualize with fluorescent microscope, most sensitive fluorescent color reaction)

  1. Wash tissue in TNT wash buffer 3 times for 10 min each at RT
  2. Incubate in Biotinly Tyramide (kit) for 60 min at RT:
    a. Dilute 1:50 with amplification reagent
    b. 80 µl biotinly tyramide, 4000 µl amplification reagent
  3. Wash tissue in TNT wash buffer 3 times for 10 min each at RT
  4. Incubate in SA-Fluorophore (FITC) for 60 min at RT
    a. Dilute FITC 1:500 in TN blocker
    b. 2 µl FITC, 1000 µl TN blocker
  5. Wash tissue in TNT wash buffer 3 times for 10 min each at RT
  6. Use FITC mounting medium and coverslip tissue
TNT wash buffer 1L
15.76 g Tris-HCl (0.1M final)
8.77 g NaCl (0.15M final)
0.5 ml Tween 20 (0.05% final)
Bring up to 1L with dH2O
pH 7.5
TN Blocker
Follow directions in NEL-700
TSA-Biotin System Kit
5X Maleate buffer 1L
58g Maleic acid in 850 ml of milli-Q H2O
pH to 7.5 using NaOH pellets
Add 43.8 g of NaCl
Bring up to 1L with milli-Q H2O

Fast Red color reaction: (uses – AP tagged probes, red precipitate and rhodamine fluorophore, visualize using light microscope or fluorescent microscope)

  1. Prepare fast red:
    a. Add 1 fast red tablet to 2 ml of 0.1M Tris-HCl/ 0.4M NaCl buffer, pH 8.2
    b. After tablet is dissolved (takes a long time), filter solution
  2. Wash embryos in 0.1M Tris-HCl/ 0.4M NaCl buffer for 30 min, 2 times
  3. Transfer embryos to a 24-well plate
  4. Add fast red solution to wells with embryos and put plate in a dark place
  5. Monitor reaction via dissecting microscope (can take up to 2 hours)
  6. When reaction is complete, transfer embryos back to tubes and remove fast red solution
  7. Rinse with 1XPBS/0.1% Tween for 10 min.
  8. Begin TSA protocol at Step #1 if double labeling

Fast Red Buffer: pH 8.2
0.1M Tris HCl – 2.9g/ 500 ml
0.4M NaCl 11.- 6g/ 500 ml

NBT/BCIP: (uses -AP tagged probes, purple precipitate, use light microscope to
visualize, most sensitive color reaction)

  1. Incubate 2 times/ 5 min each in Genius 3 and 0.1% Tween.
  2. Transfer embryos to a 24-well plate
  3. Prepare NBT/BCIP per manufacture’s instructions, incubate 0.5 to 2.0 hours at RT
  4. with NO agitation in the dark, monitor reaction frequently.
  5. Wash in Alk. Phosphate/ 0.1 % Tw wash buffer for 30 min at RT.
  6. Wash in 1X PO4 for 5 min at RT.
  7. Fix in 4% Para & PO4 for 30 min at RT
  8. Rinse 5 min in 1X PO4 / 5% Sucrose
  9. Transfer tissue to new container, store in glycerol/PBS (2:1) or 100% glycerol at 4°C
  10. in the dark or begin TSA protocol at Step #1 if double labeling

1X MaleateTw/1% DMSO/ 1% BSA buffer solution 1L
200 ml 5X Maleate
10 g BSA
10 ml DMSO
dH2O up to 1L
pH 7.5
add 1ml Tween
Store at 4°C

10X Tris/NaCl Stock (1M Tris-HCl & 1M NaCl) pH 9.5
In 350 ml of milli-Q water add 78.5 g Tris-HCl, let dissolve then add:
29 g NaCl
Use lots of NaOH pellets to bring pH to 9.5, bring volume up to 500 ml with milli-Q
Sterile filter using bottle top filter into very clean bottle

10X MgCl2Stock
In 420 ml of milli-Q water dissolve 50.8 g MgCl2
Sterile filter using bottle top filter into very clean bottle

Genius buffer 3 pH 9.5
100 ml of 10X Tris-HCl/NaCl stock in 800 ml milli-Q water
Check pH, should be 9.5
While mixing add 100 ml of MgCl2 stock (do not adjust pH at this point)

Alkaline Phosphatase/ 0.1% Tween Wash
Dissolve 1.57 g of Tris-HCl in 950 ml of milli-Q water
pH to 7.5 and then add:
0.292 g EDTA
9.0 g NaCl
1 ml Tween 20
Bring volume up to 1L with milli-Q water


10 ml of 1M Tris-HCl in 850 ml of milli-Q water, pH to 7.5

Add 2 ml of 0.5 EDTA
9 g NaCl
Bring volume up to 1L with milli-Q water