PK: We store 10mg/ml (1,000x) aliquots of Proteinase K at -20oC. If I were making stocks just for slice in situ抯 I would make 250 ml aliquots of 100x PK.

Glycine: I use ≥2 mg/ml. Typically I dump ~1g into 250 ml of PBS.

Acetic Anhydride: Many protocols call for constant stirring for this step. I find that mixing the acetic anhydride with the TEA buffer immediately prior to the incubation is sufficient and much easier. I do this by adding 625祃 of acetic anhydride to 250 ml of 0.1M TEA in a 500 ml bottle, cap and shake vigorously, pour into bucket and add slides.

TEA: Triethanolamine buffer is made with powdered triethanolamine into a 1M (10x) stock.

Hyb Buffer: This recipe is from our whole mount protocol and has not been modified for the section in situs. We make this up in quantity and store it at -20oC.

50% Formamide (high quality, deionized)

5xSSC, pH 4.5 (pH with citric acid)

50 礸/ml yeast RNA

1% SDS

50 礸/ml Heparin

Day 2

The washes can be stored in amber bottles at room temperature and re-used indefinitely. High quality, deionized formamide need not be used to make up these washes, cheaper bulk formamide is adequate.

All of the washes are poured out at once into dedicated staining dishes. The first two are microwaved for 2 minutes (1min per 250ml in our microwave brings the solution to ~65oC) while the remainder are put into a 65oC water bath to come up to temp.

50% Formamide

5x SSC, pH 4.5

1% SDS

Wash 2:

50% Formamide

2x SSC, pH 4.5

TBS: Dilute 10x to 1x and add TWEEN-20 to 1%. Make 2 liters.

1.4 M NaCl 80g

27 mM KCl 2g

250 mM Tris, pH 7.5 250 ml of 1M Tris 7.5

Mixed serum: To block I use mixed serum from as many of the animals involved in the experiment as I can. This generally includes, chick , goat, rabbit, donkey, sheep and calf serums mixed in nonspecific quantities. Blocking solution and Ab incubation solutions are mixed to the appropriate concentrations of serum (5% to block and 1% to stain) and then filtered prior to use. If serum is precious a 5% solution can be stored at -20oC, thawed filtered and re-used as block and as a stock for the 1% sol抧. You will need 250 ml of 5% block.

aDig Ab: I use the BoeringerMannheim Ab at 2500x. I do these incubations in slide mailers that require 15ml of sol抧 for every 5 slides. Typically I make up 60 ml of 1x Ab using 24祃 of Ab (enough for 20 slides). The slides can stay in these slide mailers through the subsequent washes and, if your washes are good, can also be used for the color reaction.

NTMT: This buffer will acidify and precipitate over time so it is made fresh prior to each use. Make 1 liter to wash in TissueTek buckets and 250ml to wash in slide mailers.

1 liter 250ml

100 mM NaCl 20ml of 5M NaCl 5ml

100 mM Tris, pH 9.5 100ml of 1M Tris 9.5 20ml

1% TWEEN-20 50ml of 20% TWEEN 10ml

NBT/XPhos: If more than 1 day of development is desired it is advisable to provide fresh 1x substrate each day to avoid the formation of large, ugly, insoluble crystals.

1x reaction mix: dilute stocks to 1x in NTMT

Sigma Fast Red: To get a bright red fluorescent precipitate I use SigmaFast FastRed TR/Napthol AS-MX Tablet sets, product # F4648. A good signal will give a good brightfield red color and a very sensitive fluorescent red signal. I use this stuff at 200l/slide. Most of the reaction is typically complete after ~1hr but I typically coverslip the slides and leave them overnight. This gives me better sensitivity without significant background but some minor loss of signal resolution at high mags.

Day 3

Vectastain A/B and DAB: We use Vectastain kits for these steps: Vectastain ABC Elite (#PK-6100) and Peroxidase Substrate Kit DAB (#SK-4100).

Flourescein aMus: This is a Jackson ImmunoResearch Antibody, code#: 715-095-150

Alexa488 aFlour (Rabbit) & Alexa488 aRabbit (Goat): These are from Molecular Probes Alexa488 Signal-Amplification Kit for Flourescein Conjugated Probes (A110-53). The Alexa488 moiety has very similar spectra to FITC but is supposed to be more intense and can be used with FITC filters. Wash the slides well between these steps as background will accumulate.

When Things Don抰 Work:

The first thing to replace when things don抰 work is the probe. If stored in HybBff at -20oC probes seem to be stable indefinitely. If your probe does not work the first time, redo your Tx rxn with clean solutions (DEPC) and check the product on a gel. If a probe stops working either just replace it with newly transcribed probe or try heating it to 80oC for 30?to relieve secondary structure that may have formed during storage. I have reused probes over ten times with no apparent dimunition in signal or increase in background. I simply store them at -20oC when not in use and take them out to 70oC for the hybridization.