Immunostaining

1. Humidified staining box either an adapted "sandwich" box or purpose-made black perspex box with transparent perspex lid (Genex Ltd., Coolsdon, Surrey, UK).

2. Blocking solution, either bovine serum albumin (BSA) (0.1-1%), or normal serum (1-5%) from the species in which the secondary antibody was raised, diluted in PBS.

3. Primary antibody, diluted appropriately with blocking solution.

4. Secondary antibody diluted with blocking solution (usual range 1 in 100 to 1 in 200).

5. Tertiary antibody, e.g., Extravidin-FITC (Sigma) or ABC-peroxidase (Vector Labs. Ltd.) at the recommended dilution in PBS.

6. For immunoperoxidase:

a. Suitable substrate, e.g., diaminobenzidine (DAB). For 10 mL, dissolve 6 mg DAB in 10 mL PBS and, immediately before use, add 3 pL of 30% H2O2, or a DAB alternative, e.g.,Vector VIP.

b. Suitable counterstain, e.g., for DAB, 0.1% toluidine blue.

7. For immunofluorescence:

a. An anti quenching mountant. For 100 mL, dissolve 2.5 g 1,4-diazabicyclo [2.2.2] octane (DABCO) (Sigma) in 90 mL of glycerol at 37°C, and 0.1 g sodium azide in 10 mL of PBS. Then combine, mix well, and store in a dark bottle at 4°C.

b. Nail varnish for sealing cover slips onto slides.

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