Immunocytochemistry and Immunofluorescence Step by Step Guide

                                                                                                                           Figure 3. Direct and indirect detection

                                                                                                    Advantages and limitations of direct and indirect detection

1. Single staining: the primary antibody may be raised in any host species. Select a secondary antibody directed against that host. For example, a mouse primary antibody requires an anti-mouse secondary antibody such as goat anti-mouse or donkey anti-mouse.

2. Double or multiplex staining: choose primary antibodies raised in different host species and match each with the appropriate secondary antibody. Select spectrally separated fluorescent secondary antibodies to avoid signal overlap. For three-color labeling, green, blue, and red fluorophores are commonly used to facilitate clear signal discrimination.

1. Primary-antibody incubation: select a primary antibody appropriate for the target antigen and sample. Dilute it according to the product instructions, add it to the sample, and incubate overnight at 4 °C for 12–16 h.

2. Wash: recover the primary-antibody solution and gently wash three times on a shaker with TBST or PBST for 5–10 min each to remove unbound antibody. Adjust the number and duration of washes as needed.

3. Secondary-antibody incubation: select a suitable fluorescent secondary antibody based on the primary antibody and the sample. Ensure that its fluorescence does not overlap with sample autofluorescence. Incubate for 1 h at room temperature in the dark and follow the instructions for dilution and use.

4. Wash: remove or recover the secondary-antibody solution and gently wash three times on a shaker with TBST or PBST for 5–10 min each. Adjust the number and duration of washes as needed.

·Protect the sample from light after incubation with a fluorescent secondary antibody.

·Determine an appropriate working concentration for the secondary antibody to avoid absent signal or excessive background.

·Maintain humidity in the incubation chamber and prevent sample drying.

·Use gentle agitation during washing to prevent cell detachment.

1. Place one drop of mounting medium on the slide. Slowly lower the coverslip with the cell-bearing surface facing the slide, then remove excess mounting medium with absorbent paper.

2. Gently seal the perimeter of the coverslip with clear nail polish to prevent movement during microscopy. After the seal dries, image immediately or store at 4 °C in the dark. 

·Avoid bubbles. Add mounting medium slowly and evenly and gently adjust the coverslip with forceps. Bubbles interfere with microscopic observation and reduce accuracy.

·Choose a suitable mounting medium. Common options include buffered glycerol and antifade mounting media. Base the selection on the experiment and specimen.

·Protect mounted samples from light and maintain suitable humidity to reduce fluorescence quenching and drying.

·After mounting, cell samples can theoretically be stored for one week at −20 °C or 4 °C. For clear images and high fluorescence intensity, image them as soon as possible.