A Complete Guide to Cell Coverslip Preparation

Cell coverslip preparation is a technique in which cells are cultured on glass or plastic surfaces to enable them to adhere and spread. Typically, the cell slide is immersed in culture medium, allowing cells to grow on its surface. This technique is widely used to observe cell morphology, movement, and cell-cell interactions. By performing in vitro experiments using coverslips, various in vivo interference factors can be excluded, facilitating specific interventions on cells.

In experiments such as immunofluorescence (IF), in situ hybridization (FISH), and apoptosis detection (TUNEL), high-quality coverslip preparation is essential, as it directly affects image quality and experimental data accuracy. This article outlines the steps and precautions for cell coverslip preparation to help you quickly get started.

Step-by-Step Protocol

01 Coverslip Preparation

1. Choose the cell slide size based on your experimental requirements.

2. Cell Slide Treatment with Acid: Soak the cell slides in 5% diluted hydrochloric acid overnight (immerse them individually to prevent sticking and incomplete cleaning). The next day, rinse thoroughly with tap water 20 times, followed by triple-distilled water twice to remove any residual acid.

3. Soak cell slides in anhydrous ethanol overnight to remove lipid contaminants. Store them in 75% ethanol for long-term use (autoclaving is not required). Before use, remove the cell slides with tweezers, flame them with an alcohol lamp, and place them into the well plate.

02 Cell Seeding on Coverslips

1. Harvest adherent cells and count them. Dilute the cells in complete culture medium to the desired concentration based on experimental needs.

2. Add a small amount of culture medium to the well to create surface tension for the cell slide to adhere to the plate. Carefully place the cell slide into the well (avoid air bubbles, as they may dislodge the cell slide when adding the cell suspension). Ensure aseptic technique throughout the process.

3. Seed an appropriate number of cells onto the cell slide and incubate under standard culture conditions.

03 Fixation of Coverslips

Choice of Fixative:

1) Paraformaldehyde (Commonly 4%):

Suitable for immunoelectron microscopy, immunohistochemistry, immunofluorescence, and TUNEL staining. Fix the sample at room temperature for 15-30 minutes before proceeding with subsequent experiments.

2) 4% Neutral Formaldehyde:

Also known as 10% neutral-buffered formalin, it is widely used for immunohistochemistry, immunofluorescence, and TUNEL staining. It preserves morphology well and has excellent permeability. However, prolonged storage can lead to oxidation into formic acid, reducing the solution's pH and affecting staining quality. Cross-linking during fixation may mask antigenic sites, leading to false-negative staining results. Fix at room temperature for 15-30 minutes.

3) Other Fixatives:

Examples include cold acetone or 75%-95% ethanol, depending on experimental requirements.

Fixation Protocol Using Paraformaldehyde:

1. Prepare 4% paraformaldehyde (PFA) and store at 4°C. Warm to room temperature before use.

2. Wash the cell slide with PBS 3 times for 3 minutes each, gently agitating to avoid detaching the cells.

3. Fix the cell slide with 4% paraformaldehyde for 15 minutes (protect it from light if cells exhibit autofluorescence). Wash with PBS 3 times for 3 minutes each.

4. Permeabilize cells with Triton X-100 (prepared in PBS; adjust concentration based on the experiment) for 20 minutes at room temperature.

5. Wash with PBS 3 times for 3 minutes each.

6. Proceed with downstream experimental steps as required.

04 Coverslip Mounting

Depending on the experiment, determine whether to incubate with antibodies or perform nuclear counterstaining before mounting. After the final PBS wash, use absorbent paper to dry excess liquid from the cell slide and seal it with an appropriate mounting medium.

Precautions

Use Fixed Cell Slides Promptly: Fixed cells should be used as soon as possible to prevent degradation or experimental interference.

Handle Cell Slides Carefully: Use minimal force when picking up cell slide, grasping the edges to avoid breakage or scratches.

Gentle Rinsing: After fixation, rinse cell slides gently with PBS to prevent cells from detaching. Adjust the number of rinses based on the experiment.

Coating for Poorly Adherent Cells: For cells with poor adherence, pre-coat cell slides with substances such as gelatin, poly-L-lysine, or rat-tail collagen.

Protect Fluorescent Cells: If cells are fluorescently labeled, perform all steps under dim light conditions.

Common sizes of round cell slide :

Note: Laboratory protocols may vary. The method described here has been validated through our practice.