TUNEL staining of whole Xenopus embryos - Harland protocol (Conlon lab)

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Protocol submitted by VGP from Conlon lab protocols [1]

link to protocol document [2]

TUNEL Staining of Whole Xenopus Embryos

(Based on Harland lab protocol)

Day 1:

5 Minute washes:

Abs MeOH
75% MeOH
50% MeOH
25% MeOH

Bleach approx 30 minutes in Bleaching Solution (for 5ml: 0.125 ml 20X SSC, 0.2ml hydrogen peroxide, 0.25ml formamide, 4.5ml water).

Wash 2x 15 minutes in PBS

Incubate 1 hour in TdT buffer (this is provided with the enzyme as 5X buffer, dilute with PBS). Note that the buffer supplied with the enzyme is very limited. Use an absolute minimal volume to cover the embryos- 250-300ul per vial. More can be ordered from Invitrogen: TdT enzyme (supplied with buffer) Cat # 10533-065; Extra buffer: 16314-015.

Incubate overnight at room temperature in reaction mix : TdT enzyme at 1:100- 15U per 100ul of rxn mix; DdUTP (Roche Cat #:11-093-274-910) at 1:1000 in 1X buffer.

Day 2:

Wash 2X 1Hour at 65C in 1mM EDTA in PBS

Wash 4X 1Hour in PBS

Wash 2X 5 min in 1X MAB (see in situ protocol)

Incubate 1 Hour in 2% BMB blocking solution (I generally also add 10% Serum)- see in situ protocol for recipe.

Add in 1:3000 dilution of AP-anti-DIG antibody- incubate overnight at 4C.

Day 3:

Wash 5X 1 hour in 1X MAB

Wash 2X 5 min in AP buffer (see in situ protocol) Add staining solution: 4.5ul of Nitro-Blue Tetrazolium (NBT) and 3.5ul of 5-Bromo-4-chloro-3-indolyl-phosphate (BCIP) per mL of AP buffer. Incubate at 4C (protect from light)- staining takes 15-30 minutes to come up (check after 10 min or so)

Notes about double TUNEL- in situ hybridization

- Always do the in situ first.
- It works best to do the in situ with Magenta phosphate and the TUNEL in blue (above staining).
o To do the in situ with Magenta phosphate- follow the lab insitu protocol until the staining step. Instead of BM Purple, add 8.75 ul of Magenta phosphate (20mg/ml stock in DMF) and 4.5 ul of Tetrazolium Red (50 mg/ml stock in DMF) per ml of AP buffer- add to embryos.
o Staining will take much longer to come up than standard in situs- approx 1-4 days depending on the probe used. I recommend doing the first day at room temp and monitoring- you should see some faint staining by the end of the day. You can then put the vials at 4C over the weekend (staining is usually on Friday by our in situ protocol).
o Fix and bleach as will our normal in situ protocol
o Follow the TUNEL protocol above, but you can omit the bleaching step.