ICC/IF Protocol

Adherent cell culture on coverslips

1. Sterilize glass #1.5 coverslips using ethanol or UV exposure, if necessary. The exact size of coverslip will depend on the number of wells. For 6-, 12- and 24- well plates, 25 mm, 18 mm and 12 mm coverslips, respectively, will fit.
2. Coat coverslips in poly-D-lysine, fibronectin, or other substrate to assist in cell adherence and growth.
1. For poly-D-lysine, put enough poly-D-lysine solution into each well to cover coverslip, incubate for 1 h, remove the solution, and rinse 3x with sterile water. Allow coverslips to dry completely, then store coated plates at 4°C until ready to use. Coating procedures for other substrates may vary.
3. Seed cells on coverslips at desired density, which will vary depending on cell type and experimental aims. Incubate for at least 6 hours in growth media to facilitate adherence.
4. Let cells grow to desired confluence, and administer treatments as needed for the experiment.

Fixation

5. Aspirate culture media and gently wash 3x with PBS (5 min, room temperature).
6. Fix the cells using desired fixative:
1. Incubate the cells in 4% PFA for 10 minutes at room temperature, or 20 minutes at 4°C.
2. Incubate the cells in 100% methanol, chilled to -20°C, for 5 minutes at room temperature.
7. Aspirate fixative and gently wash 3x with PBS (5 min, room temperature).

Permeabilization

8. Permeabilize samples with 0.1-0.5% Triton X-100 in PBS (5 min, 4°C).
1. Other detergents, such as Tween-20 or saponin, may preserve your cell structure and target antigen best. Triton X-100 destroys membranes so is not ideal for membrane-associated proteins.
2. Permeabilization is not necessary after methanol fixation.
9. Wash x2 with PBS.

Blocking and Immunostaining

Below outlines the procedure for indirect, simultaneous ICC/IF, which employs involves incubating samples with multiple unconjugated primary antibodies at once, followed by incubation with multiple secondary antibodies labeled with different fluorophores. For direct ICC/IF, simply omit secondary antibody steps. Antibodies can also be applied sequentially, which is useful for cross-reacting antibodies (e.g. primary antibodies from the same host species). In this case, perform incubation and wash steps for blocking, primary antibody and secondary antibody for a single antigen before moving to the next target.

10. Incubate samples with blocking buffer (e.g. 5% normal goat serum, 0.3% Triton X-100, 1x PBS) (30 min, room temperature).
1. Blocking serum should be from the same host species as the secondary antibody in indirect ICC/IF. For example, if using donkey secondary antibodies, use normal donkey serum. Conversely, using normal serum from the same species as primary antibody host will result in high background due to cross-reactivity from the secondary antibody (e.g. don’t use normal goat serum with a goat primary antibody because an anti-goat secondary will recognize them both).
11. Incubate samples with primary antibodies at appropriate dilution in blocking buffer. Use a humidified chamber and incubate samples overnight at 4°C or 2 hours at room temperature.
1. Antibodies should be derived from different host species to minimize cross-reactivity of secondary antibodies.
12. Wash samples x3 in PBS-Triton X-100 (0.1%).
13. Incubate samples with fluorochrome-conjugated secondary antibodies at appropriate dilution (typically 1:500 – 1:1000) in blocking buffer (1 h, room temperature in the dark). From this point on, light exposure should be minimized as much as possible.
14. Wash samples x3 in PBS-Triton X-100 (0.1%).

Counterstaining, Mounting, and Imaging

15. Incubate samples with 1 μg/ml DAPI (5 min, room temperature) or other counterstain.
16. Place a drop of mounting medium containing anti-fade onto a slide and carefully place an inverted coverslip (cells facing the slide) onto the mounting medium.
17. Carefully remove any excess mounting media, and seal the coverslip edges with nail polish if needed. Some mounting media will cure following exposure to air, rendering sealing unnecessary.
18. Visualize cells under a fluorescence microscope with appropriate excitation sources and filter sets.

Buffers and Reagents

Note: If the target of the antibody is phosphorylated, such as a phospho-GPCR, all buffers below (including fixatives, wash buffers, blocking buffers etc.) should include protein phosphatase inhibitors (PPIs) according to manufacturer's instructions (often 1 PPI tablet per 10 ml solution).

General

1X PBS

• 6.46 mM Na₂HPO₄.2H₂O
• 1.49 mM NaH₂PO₄.2H₂O
• 137 mM NaCl
• 2.68 mM KCl

0.1 M Phosphate buffer

• 108 mM Na₂HPO₄.7H₂O
• 25.3 mM NaH₂PO₄.H₂O

Pre-extraction buffers

Triton X-100

• 1x PBS
• 0.5% Triton X-100

Cytoskeleton (CSK) buffer

• 100 mM NaCl
• 300 mM sucrose
• 3 mM MgCl₂
• 10 mM PIPES

PTEMF buffer

• 20 mM PIPES pH 6.8
• 0.2% Triton X-100
• 10 mM EGTA
• 1 mM MgCl₂
• 4% formaldehyde

Fixatives

4% Paraformaldehyde (PFA)

• 4 g PFA powder
• 10 μl 10 N NaOH
• 100 ml 0.1 M phosphate buffer

Heat phosphate buffer in a glass container in microwave for 30 – 60s (do not boil). Transfer to a heated stir plate (~65-75°C) in a fume hood and add stir bar and NaOH. Add 4 g of PFA to the heated solution and leave stirring for several minutes until all of the PFA has dissolved (there may be a few granules left undissolved). Cool and filter, then store at 4°C.

Glyoxal solution mix (~4 ml)

• 2.835 ml ddH2O
• 0.789 ml ethanol (absolute, for analysis)
• 0.313 ml glyoxal (40% stock solution)
• 0.03 ml acetic acid

Vortex and bring to pH 5 with drops of 1 M NaOH. Store at 4°C and use within a few days. For further detail, see Richter et al. Glyoxal as an alternative fixative to formaldehyde in immunostaining and super‐resolution microscopy. EMBO J. 37, 139–159 (2018).

Methanol

• 100% Methanol (-20°C)

Methanol/Acetone

• 50% Methanol (-20°C)
• 50% Acetone (-20°C)

Blocking solutions

Serum block

• 1X PBS
• 1-5% normal donkey or goat serum (depending on host of secondary antibodies)
• 0.1-0.3% Triton X-100

BSA block

• 1X PBS
• 1% BSA
• 0.1-0.3% Triton X-100

PBT-G

• 1X PBS
• 1% BSA
• 0.05% Tween-20
• 300 mM glycine

Wash buffers

Triton X-100

• 1X PBS
• 0.1% Triton X-100

Tween-20

• 1X PBS
• 0.05% Tween-20