Blocking Reagent Protocol & Troubleshooting

Protocol

Preparation of Stock Solution (10x)

Blocking Reagent is dissolved in Maleic acid buffer [100 mM Maleic acid, 250 mM NaCl, pH 7.5 (20 °C) adjust with conc. or solid NaOH], to a final concentration of 10% (w/v) with shaking and heating either on a heating block to +60 °C for approx. 1 h, or until the reagent is completely in solution, or in a microwave oven 3 to 4 min total. The Blocking Reagent must be heated while dissolving, but must not be boiled. Boiling causes the reagent to coagulate. When the Blocking Reagent does not go into the solution, check the pH of the solution, and adjust if necessary, and continue heating.

Note: Dilute the 10x Blocking Solution with Maleic acid buffer to a 1x Blocking Solution. Always prepare freshly!

Blocking Reagent and Whole Mounts

During the in situ hybridization procedure, the nonspecific background is more efficiently reduced by adding Roche tRNA or Roche fish sperm DNA to the hybridization solution.

The Blocking Reagent is used to decrease the background in nonradioactive hybridization and detection of nucleic acid hybrids: 1) Blocking prior to the detection procedure is optional; either use the blocking reagent or the appropriate serum from the animal (e.g. sheep) from which the antibody was obtained. Antibodies may be pre-adsorbed for 1 hour using a whole-mount specimen to reduce the background.

Protocol for "Use in Immunological Detection in ISH"

For details, refer to DIG-RNA probes & tissue sections in the Roche Non-radioactive In situ Hybridization Manual.

After last washes, continue to immunological detection:

Place slides in a tray suitable for 8 slides with 30 mL of buffer. Incubate as follows (and change trays rather than just solutions and wash the extra set of trays between incubations to avoid background):

1. Buffer 1 [100mM Tris-HCl pH7.5, 20 °C; 150 mM NaCl] 5 minutes
2. Buffer 2 [buffer 1 with 0.5% (w/v) Blocking Reagent] 1 h
3. Buffer 3 [buffer 1 with 1% (w/v) BSA; 0.3% (v/v) Triton X-100] 1 hour
4. Drain excess buffer 3 (do not let the tissue become dry), and add 100 μl buffer 4 [buffer 3 with anti-DIG-AP 1:1000 to 1:3000 depending on your target) on the tissues

Prepare Freshly Before Use

5. Incubate 1 hour at RT in a moist chamber
6. Wash 4 times in buffer 3 (20 minutes)
7. Equilibrate in buffer 1 for 5 minutes
8. Incubate 5 minutes in buffer 5 (100 mM Tris-HCl (pH 9.5, 20 °C; 100 mM NaCl; 50 mM MgCl₂);
9. Perform the color reaction (e.g. NBT/BCIP) in the dark; cover trays to avoid evaporation; check in appropriate time intervals; stop enzyme reaction and wash off background.

Microarrays

In general, Roche recommends using arrays with epoxy or any other non-charged surface to keep background signals as low as possible.

Most important is appropriate blocking prior to the addition of the fluorophore-conjugated anti-DIG. Roche recommends using the blocking buffer included in the DIG Wash and Block Buffer Set.

First, wash the array after hybridization, and then pre block using a solution of 1% Blocking Reagent in 2x PBS containing 0.1% Tween 20. Dilute the dye-labeled anti-DIG in 1% Blocking Reagent in 2x PBS containing 0.1% Tween 20 and apply it to the array. For washing use 2x PBS, 0,1% Tween 20. Alternatively, a buffer containing 1% BSA instead of the DIG Blocking Reagent or 5% Denhardt 's reagent can be used.

Troubleshooting

Increased concentration, up to 5%

The concentration of blocking reagents can be increased by up to 5%. This can have a positive effect on reducing background staining. For most standard DIG applications, the recommended concentration is sufficient. For biotin detection, use a concentration of 5%.

The easiest way to use the Blocking Reagent is with the Roche DIG Wash and Block Buffer Set (Product No. 01585762001), a ready-to-use set of stock solutions.

High Background

The reason is possibly nonspecific sticking of the labeled DNA to proteins or other DNA-binding molecules in the tissue. To reduce high background staining, follow the advice given in the protocol help corner, in particular, consider pre-adsorbing the antibody mix.