Substrates

Alkaline Phosphatase development.

Three procedures are commonly used. BCIP/NBT which gives a blue stable stain, new fuschin red, which give a stable (but less sensitive) red stain, and Boehringer BM purple, a commercial ready made product that produces, you guessed it, a deep purple stain. There is also a green substrate from Boehringer, but we have not tested it. The reagents for the red stain can be easily assembled from the protocol in the book Antibodies, by Harlow and Lane (CSH Press) or you can use the kits from K&L labs, Genetics Resources and others. Notes on double staining are provided below.

Notes

In all cases levamisole should be added to 2 mM to block endogenous phosphatases. Blue stain is very stable in BB:BA. If the staining is faint, or you wish to go on and stain with a second antibody you should refix embryos after staining. Fixing can be achieved with either Bouin's overnight followed by washing in 70% methanol if you do not want to restain, or with MEMFA for 30 minutes followed by rinsing in TBST if you would like to restain. Bouins is probably best at stabilizing stain and gives a nice yellow counterstain. The red stain is NOT stable in methanol, but seems stable in BB:BA, so dehydrate and get it in there quick!

Blue. Exchange TBST for AP buffer with freshly added levamisole (2 mM-TOXIC!!!). Wash RT 5', repeat. Replace with AP buffer +lev+ 3 ul/ml 100 mg/ml NBT + 3.5 ul/ml 50 mg/ml BCIP. Develop at RT until staining obvious. Rinse with TBS, dehydrate with MeOH, mount in BA:BB (1:2). AP buffer is 100 mM Tris pH9.5, 5 mM MgCl2, 100 mM NaCl, 0.1 % tween-20. Blue stain is stable in MeOH, BA:BB and in bleach.
Red. Use the kit from Kirkgaard and Perry Labs. Exchange TBST for TBST plus 2 mM lev. Wash RT 5'. Repeat. Make up stain by adding 20 ul PhThaloRed to a tube, then adding 20 ul of activating solution, mix and stand for 3'. In another tube mix 1 ml of water with 100 ul of Buffered Substrate solution+ levamisole. Mix the contents of the two tubes and add to embryos. After about 10 minutes in the dark check staining. It is possible that the red stain is unstable in methanol, but it is very stable in BA:BB so exchange methanol for clearing soution ASAP. The red stain is NOT resistant to bleaching, and you can use this, or just methanol, to selectively remove red if overstaining occurs, or if the red is masking other staining patterns.
Purple. Exchange TBST for TBST with freshly added levamisole (2 mM-TOXIC!!!). Wash RT 5', repeat. Replace with 500 ul BM purple plus 1mM levamisole. Treat as per BCIP/NBT. Very good stain with low background, excellent for in situ hybridizations, but no real advantage for antibody stainings in our hands (BCIP/NBT is fine).

Horseradish Peroxidase

We use three methods. These all use diamino-benzidine in some form or another. This is a powerful carcinogen so use extreme caution. Wear gloves and dispose of all tips and tube with have come in contact with staining solution into 10% bleach to inactivate DAB. These stains are very stable so do not overstain, it will never leach out! Details on making your own reagents are available in Harlow and Lane, or you can buy kits. We use the Enhance kits from K&L labs (Suppliers)

Orange. per ml of staining solution mix 1.0 ml of water with 100 挈 of enhance orange buffer, plus 20 挈 DAB-C, plus 20 挈 peroxide. Develop in the dark checking at 10 minute intervals. Usually only take 10 to 20 minutes to develop. Very stable, excellent stain for second round in double stainings.
Black, this is the nickel enhanced DAB procedure. per ml of stain 1.0 ml water, 100 挈 enhance black buffer, 20挈 DAB, 20挈 peroxide. Develop in the dark check at 10' intervals.

Double (or triple) staining

There are many ways to do multiple rounds of staining with different substrates. The easiest is just to do multiple rounds. Simply stain with one antibody/substrate combination, then restain with a second antibody/substrate, using a different substrate of course. The only thing to remember is that if both antibodies are linked to the same enzyme, e.g. alkaline phosphatase, or if both primary antibodies were generated in the same species, e.g. mouse, some activity will remain from the first round and will also stain up in the second round.

There are two ways around this. The most simple (which we use) is to develop the first round with the DARKER colored substrate (e.g. blue or purple) and the second round with a light colour (e.g. red AP or orange HRP). This means that even if your blue cells stain a little with the red substrate you will not be able to see it.

This is how the double stainings of pronephros/pn duct were done.

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