Whole-Mount in situ hybridization

In Situ Protocol Checklist (Wills Lab, 3/5/19 by JHP)

Note: This protocol uses probes with digoxigenin substituted ribonucleotides (Dig-UTP).

Note: This protocol is adapted from a more detailed protocol that includes variations on incubation times (for instance, antibody exposure can be done O/N at 4C between day 2 and 3) as well as more details on how the process works.

Note: “** TIME!!” indicates a step that is time sensitive. Other steps have some degree of flexibility, but long/short incubations in these steps can damage embryos or probe.

Sample Preparation

Fix embryos in MEMFA for 1-2 hours at RT or O/N at 4°C.

Immediately transfer for methanol to wash (this is very important for embryos still in the vitelline envelope.

Embryos can then be stored at -20°C long term

Day 1 (~3 hours)

Before starting, remove an aliquot of 20% PFA from the -20°C and place in the 60°C bath

▢            100% Methanol                                                                      5 min

▢            75% Methanol, 25% H2O                                                       5 min

▢            50% Methanol, 50% H2O                                                       5 min

▢            25% Methanol, 75% PBSTw                                                    5 min

▢▢▢      PBSTw                                                                                      5 min

▢            ProtK (use 25 μL of 20 mg/mL for 50 mL PBSTw)                   5 min** TIME!!

               ProtK is kept at -20, needs to thaw. Normally keep one in use in the small fridge

▢            0.1M Triethanolamine                                                              5 min

▢            0.1M Triethanolamine                                                              5 min

▢▢         125 μL acetic anhydride in 50 mL 0.1M triethanolamine        5 min** TIME!!

               Acetic anhydride is in the chemical hood, make right before you use

▢▢         PBSTw                                                                                       5 min

▢            4% PFA (use 10 mL from -20 in 40 mL PBTw)                          20 min** TIME!!

▢▢▢▢▢ PBSTw                                                                                      5 min

▢            Prehybridize at 60°C                                                                1 hour+

               Save after for Day 2                                 

▢            Place each basket into the appropriate probe at 60°C              O/N

               Use probe at 1 μg/mL in 15 mL Falcon tube

Day 2 (~9 hours)

▢            Transfer probes back to rack and into saved prehyb at 60°C             3 min

▢▢         2x SSC at 60°C                                                                                     3 min

▢▢▢      2x SSC at 60°C                                                                                     20 min

               Recommend thawing 10 mL of BMB blocking reagent from the -20°C

▢            RNase A (use 100 μL from 10 μg/mL for 50 mL 2x SSC) at 37°C       30 min** TIME!!

▢            2x SSC                                                                                                 10 min

▢▢         0.2x SSC at 60°C                                                                                 30 min

▢▢         MAB                                                                                                     10 min

▢            BMB Blocking Reagent (10 mL to 40 mL MAB)                                   1 hour+

▢            Add 16.67 μL of anti-dig AP antibody to BMB blocking reagent       4 hours

               You can use the same block as for the previous step here

▢▢         MAB                                                                                                     5 min

▢            MAB                                                                                                     O/N

Day 3

Thaw alkaline phosphatase buffer from the -20°C

▢▢        MAB                                                                                                     10 min

▢▢        Alkaline phosphatase                                                                           5 min

▢           Place embryos in BM purple until they have developed                     ---

              Keep dark for this reaction

▢           Rinse the developed embryos with MAB                                           Short

▢           Fix in Bouin's solution at RT                                                                O/N

▢X∞?    Short washes in buffered ethanol                                                       5-10 min

▢▢▢     Long washes in buffered ethanol until not yellow                              30 min

▢           50% buffered ethanol, 50% 1x SCC                                                     5 min

▢▢        1x SCC                                                                                                  5 min

▢           Bleach                                                                                                  ~ 2 hr

              1.25 mL 20x SCC

              2.5 mL formamide

              2 mL peroxide

              45 mL water

▢▢        1x PBSTw                                                                                             5 min

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