Section8:Fixing and Staining

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1. Stop the reactions by inverting plate and dumping media into appropriate waste container. Tap gently on absorbent paper several times to remove residual liquid. For suspension cells, see note 1 below.[1]
2. Add fixative (100 ul/well). The standard fixative is 3.7% formaldehyde diluted from commercially available 37% stock solution into PBS. Alternatively, non-toxic fixatives have recently become available (See reagents below). Usually a 10-minute incubation at room temperature is sufficient, but this may vary with cell type. Other types of fixatives include glutaraldehye, methanol, etc.
3. Invert plate and dump fixative into appropriate waste container. Tap gently on absorbent paper several times to remove residual liquid.
4. Rinse 3 times with PBS + 0.1% Triton X-100 to permeabilize the cells. Incubate 5 min each time.
5. Invert, dump and blot. Add blocking buffer for one hour to block non-specific sites. We have normally used 10%FBS in PBS. At this point, plates can be stored at 4° overnight (for several days).
6. Invert, dump and blot. Add first antibody (50-100 ul/well) diluted in Blocking Buffer or PBST + 1%BSA. Incubate 2 hr at room temperature or overnight at 4°. As a guideline, use first antibody concentrations the same as or 2-fold more concentrated than the optimal immunoblot dilution. The optimal concentration will be determined by experimental design. Incubate the cells with the primary antibody for one hour at room T or overnight at 4°.
7. Invert, dump and blot. Wash 3 times with 100 ul/well PBS + 0.1% Triton (Wash Buffer). One-minute incubation is sufficient at this step.
8. Invert, dumb and blot. Add 100 ul/well of horseradish peroxidase coupled second antibody of appropriate species specificity. In our hands, a 1:1000 dilution of Amersham Pharmacia antibodies in Blocking Buffer is optimal. Incubate 1hr at room temperature. See note 8 for alternative second antibodies.[8]
9. Towards the end of the 1hr incubation, prepare the commercially available chemiluminescent substrate solution. Mix equal volumes of the luminol/enhancer and stable peroxide reagents. Protect from light while solutions equilibrate to room temperature.
10. Remove second antibody (invert, dump and blot). Wash wells 2 times with 100 ul Wash Buffer and then 3 times with 100 ul PBS. It is important to completely remove the Triton, which interferes with the peroxidase activity.[10]
11. If the experiment involves more than 1 plate, process only 1 plate at a time. Leave others in the final PBS wash. Starting with the first plate, invert, dump and blot. Add 100 ul/well of substrate solution. Wait 1 min. Read luminescence (relative light units, RLU) on standard plate reader at 0.1 second (or longer)/well.[11]
12. While first plate is being scanned, develop second plate, etc.

Notes:

[1] For suspension cells, aliquot cells into wells of a poly-D-lysine-coated plate. We normally spin the cells in the plate for 5 minutes at 1000-1500RPM and wait one hour to allow the cells to rest prior to compound addition or stimulation. The alternative method would be to proceed with the reaction after plating the cells and then stopping the reaction by spinning cells at 1000-rpm for5 min. Invert, dump, and blot. Proceed with fixation as for adherent cells. At this point the cells will be well stuck to the plate.
[8] Fluorescent secondary antibodies can also be used with detection on:
• High information/content laser scanning detection instruments are commercially available and have been used extensively for these applications.
• The use of infrared fluorescent labels can be used in the cELISA assays by using commercially available infrared imaging systems.

[10] The development time significantly influences the luminescence. Therefore, it is not practical to compare the absolute values of RLU between plates. Each plate must contain the appropriate controls, such as min and max or standard curve.
[11] One minute is a convenient reaction time. Although the absolute values of the RLU increase with time, the relative values are consistent for 5 to 10 minutes.