IF for Anti-Glypican 4 (GPC4) [IPI-mGPC4.9]

Antibody Validation via Immunofluorescence in ExpiCHO-S™ Cells

Objective:

Immunofluorescence (IF) is a powerful technique used to visualize the localization of proteins within cells using antibodies specific to the target protein. This protocol details the validation of antibodies targeting the Glypican family (1–6) using immunofluorescence in ExpiCHO-S™ cells. This protocol integrates the EVOS M7000 fluorescence imaging system into the immunofluorescence workflow, providing a streamlined process from sample preparation to image acquisition and analysis. This protocol provides a foundation for validating antibodies through immunofluorescence in ExpiCHO-S™ Cells. It may require optimization based on the specific cell type, target protein, and antibodies used.

Materials:

• Cell line: ExpiCHO-S™ Cells (Catalog No. A29127; Thermofisher)
• Primary antibodies targeting Glypican 1, 3 or 4 (five GPC antibodies for distribution to be tested: IPI-GPC1.21; IPI-GPC1.44; IPI-mGPC3.13; IPI-mGPC3.19; IPI-mGPC4.9)
• Species-specific secondary antibodies conjugated with fluorescent dyes (Alexa Fluor® 647 AffiniPure™ F(ab')₂ Fragment Goat Anti-Rabbit IgG, Fc fragment specific (min X Hu Sr Prot) (Code: 111-606-046; Jackson ImmunoResearch Laboratories)
• Reagents:
• Complete medium: DMEM medium with 10% FBS
• Normal goat serum (NGS) (Gibco, cat. no. 16210-064)
• DAPI (Thermo Fisher Scientific, cat. no. D3571)
• Fetal bovine serum (FBS; Wisent, cat. no. 080450)
• Paraformaldehyde solution, 16% w/v in water (Beantown Chemical, cat. no. 140770-10ml)
• Triton X-100 (Thermo Fisher Scientific, cat. no. BP151-500)
• Phosphate-buffered saline (PBS, 1X)
• Fixation buffer (8% Paraformaldehyde in PBS): 5 mL of 1X PBS + 5 mL of 16% PFA in water.
• Permeabilization buffer (0.1% Triton X-100 in PBS): 50 μL of Triton X-100 + 50 mL of 1X PBS. Mix gently and store at 4 °C for 1 week. (optional)
• Blocking buffer (1X PBS with 5% BSA, 5% Normal goat serum (NGS), and 0.01% Triton X-100): 47.5 mL of IF incubation buffer + 2.5 mL of NGS. Mix gently at 4 °C and use immediately.
• IF incubation buffer (1X PBS, 0.01% Triton X-100, 5% BSA): 80 mL of 1X PBS + 10 μL of Triton X-100 + 5 g of BSA. Rock gently at 4 °C until the BSA is completely dissolved. Complete at 100 mL with 1X PBS. Keep on ice and store at 4 °C for 1 week.
• DAPI: stock concentration (5 mg/mL): Dissolve 10 mg of DAPI in 2.0 mL of deionized water. Aliquot and store at -20 °C. Working concentration (5 μg/mL): 5 μL of DAPI stock concentration (5 mg/mL) + 5 mL of water. Prepare 1 mL aliquots and store at -20 °C for years. IF DAPI final solution (0.5 μg/mL DAPI in 1X PBS): Add 0.5 mL of DAPI working concentration (5 μg/mL) to 4.5 mL of 1X PBS (final 5 mL solution).


• Equipment:
• Sensoplate microplate, 96 well, PS, F-bottom, Glass bottom, Black, LID, sterile (REF: 655892; Greiner bio-one)
• EVOS M7000 fluorescence imaging system (Invitrogen)
• Humidified CO₂ incubator

Procedure:

1. Transfect and culture CHO cell lines (Day1–3):

• Transfect CHO cells with GPC plus GFP. Final will contain both untransfected and transfected cells (samples plus negative controls mixed).

2. Seeding Cells (Day 3):

• Harvest transfected CHO cells and pellet by centrifugation (3 min)
• Wash cell pellets with 1X PBS (1.25 mL)
• Resuspend cells in 1X PBS and adjust the cell concentration to 1 × 10⁶ per mL
• Seed ~1 × 10⁵ cells per well (100 μL per well) in the PLL-coated 96 well plate
• Incubate at room temperature for 30 min

3. Fixation:

• Without disturbing the plate/cells, carefully add 100 μL of fixation buffer (8% PFA)
• Fixation buffer: mix 16% PFA solution 1:1 with 1X PBS (3.5 mL + 3.5 mL)
• Incubate 15 min at 37 °C
• Wash 3x with 150 μL PBS

4. Permeabilization:

• Add 100 μL of permeabilization buffer (0.1% Triton X-100 in PBS)
• Incubate at room temperature for 10 min.
• Wash 3x with 150 μL PBS

5. Blocking:

• Block non-specific binding by incubating cells with 100 μL IF blocking buffer (1X PBS with 5% BSA, 5% Normal goat serum (NGS), and 0.01% Triton X-100)
• Incubate at room temperature for 30 min.
• Shake off blocking buffer

6. Primary Antibody Incubation (Day 3):

• Prepare the primary antibodies against Glypicans in IF blocking buffer at the appropriate concentration.
• Add 50 μL (e.g., 1 μg/mL or 2 μg/mL for IPI-GPC1.44) of primary antibody solution
• Incubate at room temperature for 1 hour.
• Wash 3x with 100 μL IF incubation buffer (1X PBS, 0.01% Triton X-100, 5% BSA) for 5 min each with shaking.

7. Secondary Antibody Incubation (Day 3)

• Add 50 μL (e.g., 1 μg/mL or 2 μg/mL for IPI-GPC1.44) of appropriate secondary antibody in IF blocking buffer (conjugated with fluorophore in the red range — e.g. Alexa Fluor 594 [red] or 647 [far red]) Alexa Fluor® 647 AffiniPure™ F(ab')₂ Fragment Goat Anti-Rabbit IgG, Fc fragment specific
• Incubate at room temperature for 1 hour in the dark.
• Wash 2x with 100 μL IF incubation buffer (1X PBS, 0.01% Triton X-100, 5% BSA), each 5 min (with gentle agitation).

8. Nuclear Staining: (Day 3/4)

• Incubate cells with 50 μL of 0.5 μg/mL DAPI in 1X PBS for 5 minutes at room temperature.
• Wash 3x with 100 μL PBS.
• Add 50 μL PBS, then cover plate.

9. Imaging Wells using EVOS M7000 microscope (Day 3 or 4)

• Select the correct objective lens (10X) for imaging, adjust magnification (10X objective) and exposure to optimize fluorescence signal.
• Choose the corresponding fluorescence channels for the dyes used in the experiment (tgBFP for DAPI nuclear staining, GFP for green fluorescence, CY5 for red fluorescence).
• Image properties: Microns per pixel: 0.507