- Detection buffer: 4x SSC (dilute from 20x SSC, Appendix 1) containing 0.2% (v/v) Tween 20 .
- BSA block: 5% (w/v) BSA (bovine serum albumin) in detection buffer (Note 1).
- Detection solution: select as required (see Figure 9.2 for explanation). Different detection reagents can be combined to detect different labels simultaneously
(i) For digoxigenin-labeled probes: anti-digoxigenin (FAB fragment) conjugated to fluorescein or rhodamine (200 m g/ml; Roche), final concentration 2-4 m g/ml in BSA block (Note 2).
(ii) For biotin-labeled probes: avidin, streptavidin or extra-avidin conjugated to fluorescein, Texas Red, Cy3, Alexa fluorphores or others (e.g. Vector Laboratories, Sigma, Molecular Probes), final concentration 5-10 m g/ml in BSA block (Note 3).
Preparations labeled with biotin, digoxigenin or other label to be detected following stringent washing (Protocol 9.1) or in situ
primer extension (Protocol 8.5)
For the washing steps (1 and 5), slides are put into staining jars and covered totally with solution. Agitation by gently moving the staining jars by hand (once every minute) or continuously on a shaking platform is recommended. To change solutions, pour off carefully and replace slowly with next solution, or transfer slides to another staining jar with the next solution. It is very important that slides do not dry out between Steps. Procedures using fluorophores should be carried out in subdued light to avoid degrading the molecules.
- Place slides in detection buffer and leave in buffer for 5 min
- Apply 200 m l BSA block to the marked area of each slide and cover with a large plastic coverslip. Incubate at room temperature or 37 ° C for 5-30 min in a humid chamber (Note 3).
- Decide which detection reagents (see Figure 9.1) are required for which slides. Calculate and make up detection solutions: For example, for 8 slides, each labeled with biotin and digoxigenin, 3 m l FITC-anti-digoxigenin (200 m g/ml, Roche), and 1.5 m l Cy3-Streptavidin (1mg/ml, Sigma) in 300 m l BSA block will be suitable (see also Note 3).
- Put the coverslip to one side and drain BSA block away. Add 30-40 m l of detection solution. Replace the coverslip and incubate for 1 h at 37 ° C in a humid chamber
- Remove coverslips and wash in detection buffer at 42 ° C for 20 min exchanging the solution three times
- Proceed to staining (Protocol 9.3)
- Numerous qualities of BSA, bovine serum albumin (50 from Sigma alone) are available; a mid-priced fraction V, essentially globulin-free, specified for use in molecular biology and immunocytochemistry protocols is suitable, such as Sigma A7638, A3803 or B4287. BSA solutions must not be autoclaved as the protein coagulates
- See Figure 9.2 for explanation. Some researchers use anti-digoxin from Sigma. Reagents to detect biotin are numerous and are available conjugated to many fluorophores (see Table 4.1).
- These are typically 1:50 to 1:500 dilutions of the stock solutions supplied by manufacturers. Concentrations must be optimized and vary with antibody batches; two-fold concentration changes are used during optimization
- This steps blocks non-specific sites that could bind detection reagents. Longer incubation at 37 ° C will result in better blocking, but is normally not necessary
- Based on Heslop-Harrison et al. 1991