IF/ICC antibody titration experiment

This protocol outlines an experiment to titrate antibodies for optimal concentration, an essential first step when testing a validated antibody for Immunofluorescence (IF)/Immunocytochemistry (ICC).

We will use NLS-GFP transfected PC3 cells that express GFP in the nucleus. 

graph LR classDef default fill:#FFF,stroke:#333,stroke-width:1px; classDef washing fill:#CBC3E3,stroke:#333,stroke-width:2px,rx:10px,ry:10px; classDef main fill:#FFB347,stroke:#333,stroke-width:2px; subgraph IF/ICC A[Fixation
10 min @RT]:::main B{{Permeabilization
15 min @RT}}:::main C[/Blocking
1h @RT/]:::main D[(Primary Antibody
overnight @4°C)]:::main E[(Secondary Antibody
1h @RT)]:::main F(((Imaging))):::main end A --> W1[Washing]:::washing W1 --> B B --> W2[Washing]:::washing W2 --> C C --> W3[Washing]:::washing W3 --> D D --> W4[Washing]:::washing W4 --> E E --> W5[Washing]:::washing W5 --> F


🚛 Reagents needed 📦 :
Item Item article number Distributor  Vendor
1⨉ PBS
Stock room Stock room
10% Triton X100
Tween 20
 Merck Sigma Aldrich
Sterile water
Stock room Stock room
BSA A9647-10G Merck Sigma Aldrich
Goat serum 
(or other sera matching the host of the secondary antibody)		 GOA-1B Capricorn Scientific GmbH Capricorn Scientific GmbH
16% Formaldehyde (w/v), Methanol-free 28908 Thermo Scientific Thermo Scientific
Buffers and working solutions

1️⃣ The following table describes the steps to prepare 0.1% PBS-Tween 20 solution.

Reagent Volume Final Concentration
PBS 50 mL --
Tween 20 50 µL 0.1%
TOTAL 50 mL --

2️⃣ Make a stock of 10% BSA (w/vol) in 1x PBS and store in 4°C.

Reagent Volume Final Concentration
1x PBS 4.5 mL --
BSA (powder) 0.5 g 10% (w/vol)
TOTAL 5 mL --

The powder can take some time to dissolve. Pipette mix and if there are still residues remaining place it on a rotator.

3️⃣Permeabilization buffer PBS-Triton 0.25%:

Reagent Volume Final concentration
1x PBS 48.75 mL
10% Triton-X100 1.25 mL 0.25% (vol/vol)
TOTAL: 50 mL


3️⃣4% (w/v) PFA in PBS:

Reagent Volume Final concentration
1x PBS 40 mL
16% formaldehyde 10 mL 4% (w/v)
TOTAL: 50 mL

Aliquot in 2 mL tubes and store in freezer. Do not freeze thaw, use and throw after thawing.

4️⃣ 1x PBS-Tween 0.1% + 1% BSA:

Reagent Volume Final concentration
1x PBS-Tween 0.1% 900 µL
10% BSA 100 µL 1% (w/vol) 
TOTAL: 1000 µL

To be used as buffer for antibody incubations steps. Store in 4°C.

Fixation

1️⃣ Aspirate and fixate by incubating with 4% PFA in PBS for 10 min.

2️⃣ Wash 3 times in PBST.

Permeabilization

Permeabilize by incubating with PBS-Triton 0.25% for 10-20 min at room temperature.

Wash 3 times in 1xPBS @5 min each.

Blocking

1️⃣The blocking step helps prevent non-specific binding by occupying potential non-specific sites on the cell surface and within the cell. This reduces background fluorescence and ensures that the detected signal comes from the specific interaction between the antibody and its target antigen. Additionally, the goat serum blocks Fc receptors on the cells, preventing non-specific binding of the secondary antibodies through their Fc regions.

Reagent Volume Final concentration
1x PBS-Tween 0.1% 180 µL
Goat serum 20 µL 10% (vol/vol)
TOTAL: 200 µL

Incubate for 1h in room temperature.

2️⃣ Wash 3x in PBS-Tween for @5 min each.

Primary antibody incubation

1️⃣  Before preparing each reaction make some working solutions of each antibody.
Make sure to thaw antibodies on ice and make all the solutions on ice.



🟣Anti-MAX 1:20,000 Working Solution

  • Prepare 1:1,000 Intermediate Dilution:
    • Mix 1 µL of Max stock with 999 µL of PBST-BSA mix to make 1,000 µL of 1:1,000 solution.
  • Prepare 1:20,000 Working Solution from 1:1,000 Dilution:
    • Mix 20 µL of 1:1,000 solution with 380 µL of BSA mix to make 400 µL of 1:20,000 working solution.

🟢Anti-GFP 1:100,000 Working Solution

  • Prepare 1:1,000 Intermediate Dilution:
    • Mix 1 µL of Anti-GFP stock with 999 µL of PBT-BSA mix to make 1,000 µL of 1:1,000 solution.
  • Prepare 1:100,000 Working Solution from 1:1,000 Dilution:
    • Mix 4 µL of 1:1,000 solution with 396 µL of PBT-BSA mix to make 400 µL of 1:100,000 working solution.

🔴Anti-cMyc 1:500 Working Solution

Mix 1 µL of Anti-cMyc stock with 499 µL of BSA mix to make 500 µL of 1:500 working solution.

2️⃣ Prepare reaction mixtures on ice and incubate in cold room overnight:

Anti-MAX Dilutions
Final Concentration Volume of 1:20,000 Working Solution Volume of PBS-Tween 0.1%, 1%BSA
1:20,000 200 µL 0 µL
1:40,000 100 µL of 1:20,000 solution 100 µL
1:80,000 50 µL of 1:20,000 solution 150 µL
1:160,000 25 µL of 1:20,000 solution 175 µL
Anti-GFP Dilutions
Final Concentration Volume of 1:100,000 Working Solution Volume of PBS-Tween 0.1%, 1%BSA
1:100,000 200 µL 0 µL
1:200,000 100 µL of 1:100,000 solution 100 µL
1:400,000 50 µL of 1:100,000 solution 150 µL
1:800,000 25 µL of 1:100,000 solution 175 µL
Anti-cMyc Dilutions
Final Concentration Volume of 1:500 Working Solution Volume of PBS-Tween 0.1%, 1%BSA
1:500 200 µL 0 µL
1:1,000 100 µL of 1:500 solution 100 µL
1:2,000 50 µL of 1:500 solution 150 µL
1:4,000 25 µL of 1:500 solution 175 µL

2️⃣ Incubate overnight on slow shaker in cold room. 🛌🌌

3️⃣  In the morning wash away the primary antibody with 3x washes in PBS-Tween for @5 min each.

Secondary antibody

Use secondary antibody that have as target the same specie and isotype as the primary antibody is raised in. In this case the primary antibodies are all raised in rabbit so we'll use Alexa Flour 647 Goat anti-Rabbit IgG (H+L) a secondary antibody raised in goat with rabbit IgG as target antigen.

1️⃣   A common concentration for secondary antibodies are 1:1000, but this step could also be optimized, especially with the blocking step.

Reagent Volume Final concentration
1x PBS-Tween 0.1% + 1% BSA 999 µL
Secondary antibody 
Alexa Flour 647 Goat anti-Rabbit IgG (H+L) 		 1 µL 1:1000
TOTAL: 1000 µL

2️⃣ Incubate with secondary antibody in room temperature for 1h.

3️⃣ 3x washes in PBS-Tween for @5 min each.

4️⃣After last wash leave cells in 1xPBS and proceed to imaging. Store samples wrapped in parafilm in 4°C.